One of several Alchemical Laboratories that operate within the influence and city limits of Central City, Laboratory 3 is one of the closest of such facilities to the Central Command station and the Führer's official residence. As described early in the series by Edward Elric, Laboratory 3 does not seem to have any significant experiments under operation, but this unassuming lab hides a rather dark secret: beneath Lab 3, in a sealed-off area, lies the entrance to a secret facility in which untold human experiments were conducted as well as a tunnel that leads directly to the inner sanctum of Father and the other Homunculi.

Though mentioned during the Laboratory 5 arc, Laboratory 3 is first seen during the "Fishing" Mission orchestrated by Colonel Roy Mustang. After the body of Barry the Chopper is chased away from Warrant Officer Vato Falman's safehouse, Barry (in his armor vessel), Mustang, Lt. Riza Hawkeye, 2nd Lt. Jean Havoc and Alphonse Elric follow it into the depths of Lab 3, where the team splits off in different directions to find clues. While Barry, Hawkeye and Alphonse make it to the very doors of Father's lair, Mustang and Havoc encounter Lust in the human experimentation lab, to devastating results.

When Edward Elric returns to investigate Lab 3 according to Al's instructions, he notices that the entrance to the underground area - previously sealed off by a locked gate - has now been completely covered up with alchemy.

Laboratory 3 appears again in Chapter 90, when Edward, Scar, Van Hohenheim, Lan Fan, Darius, Jerso and Zampano all storm the hidden entrance to the underground tunnel, intent on a final showdown with the enemy. Mustang and Hawkeye follow the same route in Chapter 93 and in Chapter 100, it is revealed by the Alchemist doctor that Lab 3 is also the location of a Five-point circle similar to that hidden in Lab 5. In fact, Edward correctly deduces that all five Central Alchemical Laboratories contain these circle and are, themselves, arranged in a five-point circle around Central Command, forming yet another layer of the increasingly complex Nationwide Transmutation Circle.