Murine cytomegalovirus replicated in reproductive tissue of male mice infected with the virus. We examined three strains of mice latently infected by injection at birth with 100 plaque-forming units of the virus. As adults, these mice contained within their testes 4--6 viral genomic equivalents per 100 cells, as tested by hybridization between mouse DNA and cytomegalovirus DNA. Acutely infected male adult CBA mice homozygous for the nude gene (athymic: nude/nude) produced infectious virus in their testes, the amounts of which varied according to the animal's age at the time of infection. Heterozygous (nude/+) litter mates contained significantly less virus than nude/nude mice. At the peak of virus replication hybridization between virus DNA and mouse DNA indicated the presence of 3.3 viral genome equivalents per testicular cell. Both in situ hybridization studies and phenol emulsion reassociation of virus DNA to DNA from purified spermatozoa localized this viral DNA to immature and mature sperm cells. Hence, murine cytomegalovirus can be harbored in testes during both acute and latent infections and can replicate in male germ-line cells.