Three contenders: Master of Orion 2, UFO: Enemy Unknown and Frontier: Elite 2.
The first remains the greatest space-based 4x ever made, so much so that it is still the standard of comparison by reviewers for every new entry in the genre. With relatively few tech options, small galaxies, a single-page backstory and a handful of races known only from a graphic and names pilfered from popular sci-fi, MOO 2's simplicity somehow managed to give a much greater sense of immersion in its universe than any other 4x I've played (Civ games included).
The second was truly original, and until its recent 're-imagining' was only imitated, not improved upon, and justifiably routinely tops polls, or comes close, of Best Computer Game Ever. With the technology of the early '90s, the sheer depth of then-novel features it managed to pack into its tactical maps - destructible terrain, multi-level battlefields, soldier psychology, burning debris, smoke inhalation and fire damage, medikits, ammo limitations, procedurally-generated maps, night battles, stunning enemies for interrogation, ransacking bodies for equipment - was extraordinary, even though in retrospect it lacked tactical depth. For sense of immersion its campaign remains unparallelled (a common, and justified, complaint about the latest entry being that, for all the effort it spends in making soldiers individual and investing the player in the characters, the campaign itself is highly non-immersive).
The third is still probably the largest game universe ever created, and a truly free-form game that allowed you to play however you liked (within the limits of its mechanics, which revolved around trading and combat). Imitators remain much less flexible, typically pay less attention to plausible physics or realistically-constructed game universes, have much less immersive and usually more restricted environments (such as EVE, whose "universe" is a sequence of relatively few wallpaper-like backdrops with very little freedom of movement within any given "room") and are usually shorter on options than Frontier's large selection of ships.
