The murine leukemia virus (MuLV)-related retroviruses are one of seven genera which together constitute the family Retroviridae. They are widespread as both endogenous and exogenous agents within vertebrates and have been associated with a variety of malignancies and other disorders. We isolated and characterized 12 endogenous representatives of this genus from a number of mammalian hosts. Subsequent sequence analysis revealed that the isolated viruses cluster into two clearly distinct groups. All of the exogenous MuLV-related retroviruses which have been isolated to date, as well as several endogenous examples, fall into the first group, whereas the second group is represented solely by endogenous representatives, including human endogenous retrovirus type E (HERV.E). The two groups are widespread within mammals, with both often present within one animal species. Despite this, there is no evidence to date that recombination between members of the different groups has occurred. Genetic distances and several other properties of the HERV.E genome suggest that if exogenous members of this subgroup exist, they are likely to have biological properties different from those of the other exogenous viruses of this genus. Several of these viruses are known to have been integrated within their hosts' genomes for a long period of time, and a most recent divergence date for the MuLV and HERV.E subgroups can thus be proposed. This date, approximately 30 million years ago, is the most recent date possible, and it is probable that the actual period of time since their divergence is significantly longer.