To study the possible role of immune selection in the in vivo generation of pathogenic recombinant murine leukemia viruses (MuLV), we have constructed recombinant vaccinia viruses (rVV) expressing the envelope genes of three MuLV: AKR623, MCF247, and MCF13. rVV expressing either AKR623 or MCF247 env could prime H-2b mice for anti-AKR/Gross virus CTL responses, and stimulate the in vitro generation of CTL from the spleens of mice immunized with an AKR/Gross virus-positive lymphoma. MC57 (H-2b) cells infected with either 623EnvVac or 247EnvVac could serve as targets for ARK/Gross virus-specific CTL. Cells infected with the rVV expressing MCF13 env, however, were lysed much less efficiently by these CTL. 13EnvVac was also ineffective in priming or stimulating retrovirus-specific CTL. Finally, experiments with synthetic peptides and minigenes suggested that the reduced immunogenicity of the MCF13 envelope protein likely resulted from a single amino acid substitution within an immunodominant epitope of the p15E (TM) protein. The region of MCF13 env that encodes this epitope is derived from an endogenous xenotropic virus, while the allelic sequences in MCF247 are of ecotropic virus origin. These results suggest the potential for recombination within the MuLV envelope gene to allow escape from host cellular immune responses.