In the early 1960s, the National Institute of Mental Health (NIMH) acquired property in a rural area outside Poolesville, Maryland. The facility that was built on this property housed several research projects, including those headed by Calhoun. It was here that his most famous experiment, the mouse universe, was created. In July 1968 four pairs of mice were introduced into the habitat. The habitat was a 9-foot (2.7 m) square metal pen with 4.5-foot-high (1.4 m) sides. Each side had four groups of four vertical, wire mesh "tunnels." The "tunnels" gave access to nesting boxes, food hoppers, and water dispensers. There was no shortage of food or water or nesting material. There were no predators. The only adversity was the limit on space.
Initially the population grew rapidly, doubling every 55 days. The population reached 620 by day 315, after which the population growth dropped markedly, doubling only every 145 days. The last surviving birth was on day 600, bringing the total population to a mere 2200 mice, even though the experiment setup allowed for as many as 3840 mice in terms of nesting space. This period between day 315 and day 600 saw a breakdown in social structure and in normal social behavior. Among the aberrations in behavior were the following: expulsion of young before weaning was complete, wounding of young, increase in homosexual behavior, inability of dominant males to maintain the defense of their territory and females, aggressive behavior of females, passivity of non-dominant males with increased attacks on each other which were not defended against. After day 600, the social breakdown continued and the population declined toward extinction. During this period females ceased to reproduce. Their male counterparts withdrew completely, never engaging in courtship or fighting. They ate, drank, slept, and groomed themselves – all solitary pursuits. Sleek, healthy coats and an absence of scars characterized these males. They were dubbed "the beautiful ones." Breeding never resumed and behavior patterns were permanently changed.
The conclusions drawn from this experiment were that when all available space is taken and all social roles filled, competition and the stresses experienced by the individuals will result in a total breakdown in complex social behaviors, ultimately resulting in the demise of the population.
Calhoun saw the fate of the population of mice as a metaphor for the potential fate of man. He characterized the social breakdown as a "second death,” with reference to the “second death” mentioned in the Biblical book of Revelation 2:11. His study has been cited by writers such as Bill Perkins as a warning of the dangers of the living in an "increasingly crowded and impersonal world."