We have constructed a series of "synthetic" target cell lines for an analysis of the specificity of anti-Friend virus (FV) CTL. Our results show that murine H-2 genes and individual retroviral genes can be stable expressed in Fisher rat embryo (FRE) cells, and that their products have the potential to form target structures recognized by mouse CTL. Cells expressing H-2Db and either the env or gag genes of one component of FV, helper Friend murine leukemia virus (FMuLV), were lysed by anti-FV CTL and by CTL generated against FMuLV alone. Experiments with Db-transfected FRE clones infected only with the replication-defective spleen focus-forming virus (SFFV) component of FV indicate that the SFFV genome also provides specificities recognized by both anti-FV and anti-FMuLV CTL, thus demonstrating the existence of a crossreactive CTL population. An unexpected finding was that anti-FMuLV CTL, but not anti-FV CTL were also able to lyse FRE clones that expressed H-2Kb in either the presence or absence of FV. The use of heterologous cell lines for the construction of synthetic target cells thus offers a useful approach for the analysis of T cell specificity.