Special Agent Mulder believes in UFOs and a government conspiracy to hide or deny the truth of their existence. With his FBI partner Dana Scully, the two work in the X-Files office, which was concerned with cases called "X-Files," cases with particularly mysterious or possibly supernatural circumstances that were left unsolved and shelved by the FBI. Mulder considers the X-Files and the truth behind the supposed conspiracy the main purpose of his life.

She is an FBI Special Agent, partnered on the X-Files with Special Agent Fox Mulder. They work out of a cramped basement office at FBI headquarters to investigate unsolved cases labeled "X-Files". In contrast to Fox Mulder's credulous "believer" character, Scully is the skeptic, choosing to base her beliefs on what science can prove.

Skinner is an Assistant Director of the FBI and is the direct supervisor of Special Agents Mulder and Scully and the X-Files office. He has proved to be an ally of the pair but also has blocked their investigations on many occasions.

The Cigarette Smoking Man oversaw Scully's initial briefing on her assignment to the X-Files to debunk the work of FBI Special Agent Fox Mulder, as well as her debriefing. The Cigarette Smoking Man also stole the evidence obtained by Mulder and Scully during the show's Pilot episode, and moved it to a secret, underground storage room at the Pentagon. Although uttering only four audible words in the entire 1st season of the show, CSM eventually developed into the series' primary antagonist. The character is most commonly referred to by this description because he is almost always seen chain-smoking Morley cigarettes, and also because like other villains on the show, he has no known real name.

Made up of three key members Richard "Ringo" Langly, Melvin Frohike, John Fitzgerald Byers. Described as counterculture patriots, they were ardent conspiracy theorists, government watchdogs, and computer hackers who frequently assisted central X-Files characters Mulder and Scully, though they sometimes had their own adventures. The Lone Gunmen authored a news publication called The Magic Bullet Newsletter (a pejorative reference to the single bullet theory and, like the group's name, a reference to the Kennedy assassination), later renamed The Lone Gunman, of which Mulder was a loyal subscriber. None of them had day jobs; they relied on financial backers who believed in their cause, and what revenue the subscriptions to their paper generated. They shared a loft apartment (where they also worked) and used a 1970 VW Transporter (minibus) to commute.

Deep Throat first appears in the episode "Deep Throat", warning agent Fox Mulder of the danger he is in, and later offering to help him. He subsequently becomes Mulder's covert information source. During the first season of The X-Files, the paternal-like Deep Throat provided Mulder and Scully with information they would have been otherwise unable to obtain, leading to some interesting investigations. Being a member of the then unseen Syndicate, he was in a position to know a great deal of information. Generally, Deep Throat used his power to help Mulder, though without jeopardizing his own security, and so his information was sometimes vague and needed to be decoded by Mulder. Deep Throat felt that the truth that the Syndicate kept secret from the public needed to be known, and he expressed to Mulder that he believed Mulder was the one capable of doing so. However, Deep Throat had at least once provided Mulder with false information in order to divert him, later explaining that he believed the public was just not ready to know some truths. During the Vietnam War, and while still part of the Syndicate, he worked for the CIA. A UFO was spotted over Hanoi by the US Marines, who shot it down. The surviving EBE was brought to Deep Throat, who following Syndicate policy, executed the alien. He later recounted this to Mulder and explained that his helping Mulder is his way of clearing his own conscience after that haunting experience and to atone for what he had done. He has also stated that he was "a participant in some of the most insidious lies and witness to deeds that no crazed man could imagine". He became disillusioned with the Syndicate, though unlike Bill Mulder or Alvin Kurtzweil, he remained a member. In the first season finale of The X-Files, The Erlenmeyer Flask, Mulder was taken hostage by a group of Men in Black operatives, following his investigation into an alien-human hybrid program. Fearing for Mulder's life, Deep Throat secured access for Scully to enter a high containment facility, where she managed to secretly remove a cryogenically preserved alien fetus, which would then be used as collateral for Mulder's life. In the subsequent meeting between the operatives and Deep Throat, who would not allow Scully to make the exchange, he was gunned down by a Syndicate assassin, the Crew Cut Man, on orders from the Cigarette Smoking Man. He was buried at the Arlington National Cemetery.