The outcome of infection by lymphocytic choriomeningitis virus (LCMV) in the natural murine host is determined in large part by the cytotoxic T lymphocyte response (CTL) mounted by the host. In order to define the specificities of CTL induced by LCMV infection, we have cloned and expressed the full-length nucleoprotein (NP) gene and 75% of the glycoprotein (GP) gene of LCMV in vaccinia virus vectors and have used these recombinant viruses to sensitize syngeneic target cells to lysis by anti-LCMV CTL. We have studied the anti-LCMV CTL responses induced on three different mouse H2 (major histocompatibility complex) backgrounds. First, we find that the relative recognition of the two LCMV proteins differs markedly on different H2 haplotypes; both proteins are seen on the H2bb background, while only NP is recognized on two other haplotypes (H2dd and H2qq). Second, we show that on the H2bb background the anti-GP CTL response comprises a major component of the overall CTL response, in marked contrast to several other viruses, e.g., influenza virus, vesicular stomatitis virus, and respiratory syncytial virus where anti-GP responses, if present, comprise only a minor portion of the whole. Third, LCMV GP can be a major target antigen for CTL induced by a serotypically distinct strain of LCMV, again in contrast to the above virus systems in which CTL cross-reactivity among different serotypes is dependent largely on the recognition of "internal" proteins.