The deer mouse (Peromyscus maniculatus) is a natural reservoir for several human pathogens, but little is known about the mechanisms by which such pathogens are maintained in nature. As a first step toward developing a colony of deer mice that were permissive for infection with Sin Nombre (SN) hantavirus, we collected 68 wild P. maniculatus rufinus from central New Mexico. Mice from this cohort were used to establish 26 breeding pairs, of which 85% were fertile. In subsequent generations, fertility decreased slightly to 73% (N = 59) in laboratory-reared F1 and F2 pairs. Wild-caught females delivered 7.2 litters on average (range, 1 to 18), whereas laboratory-reared pairs delivered 5.5 (range, 1 to 13). The average time between pairing and first litter was 106 days for wild-caught animals, whereas that for laboratory-reared pairs was 71 days. None of the pairs displayed a seasonal breeding preference. Cannibalistic behavior increased from 5% in founders to 26% in laboratory-reared pairs. Mean litter size for wild-caught females was 4.3, whereas that for laboratory-reared dams was 4. Founding animals have been maintained in captivity for longer than 2 years, with only 2 deaths (4.8%). Our colony is competent for infection with SN virus. Thus, it should be useful for testing of models for maintenance of SN virus in wild rodents, and other aspects of the virus-host relationship.