Endogenous retroviruses (ERVs) are widespread in vertebrate genomes and have been loosely grouped into "classes" on the basis of their phylogenetic relatedness to the established genera of exogenous retroviruses. Four of these genera-the lentiviruses, alpharetroviruses, betaretroviruses, and deltaretroviruses-form a well-supported clade in retroviral phylogenies, and ERVs that group with these genera have been termed class II ERVs. We used PCR amplification and sequencing of retroviral fragments from more than 130 vertebrate taxa to investigate the evolution of the class II retroviruses in detail. We confirm that class II retroviruses are largely confined to mammalian and avian hosts and provide evidence for a major novel group of avian retroviruses, and we identify additional members of both the alpha-and the betaretrovirus genera. Phylogenetic analyses demonstrated that the avian and mammalian viruses form distinct monophyletic groups, implying that interclass transmission has occurred only rarely during the evolution of the class II retroviruses. In contrast to previous reports, the lentiviruses clustered as sister taxa to several endogenous retroviruses derived from rodents and insectivores. This topology was further supported by the shared loss of both the class II PR-Pol frameshift site and the class II retrovirus G-patch domain.Retroviruses (family Retroviridae) are characterized by a unique replication strategy. The RNA genome of an extracellular retrovirus is first copied into DNA by virus-encoded reverse transcriptase (RT) and is then integrated into the nuclear DNA of the host cell (35). Integration is highly stable and, consequently, infection of germ line cells can lead to vertical transmission of retroviruses from parent to offspring as Mendelian alleles (8). These retroviruses are termed endogenous (to distinguish them from their horizontally transmitted, exogenous counterparts), and they have been identified in almost all vertebrate orders examined (8, 16). Some endogenous retroviruses (ERVs) represent endogenized copies of extant exogenous retroviruses, but the majority are very old and appear to lack closely related exogenous counterparts (8,16). Analysis of these ERVs in the genomes of humans, mice, and other species indicates a longstanding association between retroviruses and vertebrates, probably dating back several hundred million years, during which retroviruses have repeatedly colonized host genomes (12,19,20,23).Most ERVs show clear homology to one another and to modern exogenous retroviruses, especially across the RT gene, which is relatively refractory to nonsynonymous substitution. Diverse retrovirus sequences can therefore be aligned in order to investigate phylogenetic relationships, and this has been instrumental in the classification of exogenous retroviruses into seven genera (alpha-, beta-, gamma-, delta-, and epsilonretroviruses; lentivirus; and spumavirus) (12,26,34,37). Although many ERVs have not been assigned to particular genera, there is a growing tendency to group them ...