This study was initiated to evaluate the in vivo infectivity and pathogenicity of a group of recombinant feline leukemia viruses (rFeLVs) previously generated by in vitro forced recombination between a FeLV subgroup A virus (FeLV-A) and an endogenous FeLV (enFeLV) envelope (env) element (Sheets et al., 1992, Virology 190, 849-855). To determine infectivity of rFeLVs, neonatal cats were inoculated with rFeLVs alone or in combination with FeLV-A. The recombinant viruses were able to replicate efficiently in vivo only when administered along with FeLV-A. Of six co-infected cats, three developed thymic lymphosarcomas, one severe aplastic anemia, and two cachexia and depression; all were viremic and seroconverted shortly after inoculation. While both virus types were detected in virtually all tissues examined from these tumor-bearing cats, there was a particularly noteworthy sequence reversion in the rFeLVs. It is known that exogenous FeLV isolates carry a conserved neutralizing MGPNL epitope in the middle of the surface glycoprotein domain of the env gene. In contrast, the parental recombinant viruses used to inoculate these cats harbored the enFeLV-derived MGPNP sequence at this position. However, all in vivo-propagated recombinants displayed the MGPNL sequence, while the env-encoded backbone flanking the MGPNL sequence was that of the parental recombinant virus. These results suggest that viruses with the MGPNL epitope have an in vivo proliferative advantage. The data also provide an explanation for the conservation of this epitope in exogenous FeLVs despite the existence of variant forms in enFeLV proviral elements with which they can recombine.