Simple retroviruses present a unique opportunity for examining the host-virus relationship. Following exogenous infection and integration into the germ line, copies of these viruses can become fixed within the genome. The resulting endogenous proviral "fossils" represent a record of past retroviral infections and forms. Previous work in our laboratory has been directed at dissecting the extensive nonecotropic murine leukemia virus content of the mouse genome. One such provirus, hortulanus endogenous murine leukemia virus (HEMV), found in a single copy in the genome of Mus spicilegus, was remarkable for characteristics that suggested that it was ancient and related to the hypothetical common ancestor of murine leukemia viruses (MLVs) and other gammaretroviral species. In the present study, we have analyzed its functional properties. Transfection of a molecular clone of the HEMV provirus into mouse-derived cell lines revealed that it is replication competent. Furthermore, host range and interference studies revealed a strictly ecotropic host range and the use of a receptor distinct from those used by other classical MLVs. The identity of nucleotide sequence of the long terminal repeats (LTRs) further suggested that HEMV is a relatively recent insertion into the M. spicilegus genome at the distal end of chromosome 7. Although unique to M. spicilegus, its presence in a homozygous state in three individuals obtained from different regions implies that it has been present long enough to become fixed in this species. Exhaustive phylogenetic analysis of all regions of the HEMV genome supported the previously assigned ancestral position of HEMV relative to other MLV-related viruses. Thus, HEMV is a relatively recent introduction into the Mus germ line but is representative of a relatively ancestral MLV group.