AKR/Gross leukemia virus-induced tumor reactive cytotoxic T lymphocyte (CTL) clones were derived from C57BL/6 spleen cells. Analysis of their specificity pattern was performed by using a panel of target cells such as E male G2 and AKR.H-2bSL1 (susceptible tumors to polyclonal anti-AKR/Gross virus CTL), and cl. 18-5 and cl. 18-12 (insusceptible variant sublines derived from AKR.H-2bSL1). Several of these CTL clones were selected for further study. Lysis of Gross cell surface antigen-positive tumor cells by these clones was restricted by the H-2Kb molecule. The cell surface phenotype of these clones was Thy-1.2+, Lyt-2.2+, L3T4-, a phenotype consistent with that of polyclonal anti-AKR/Gross CTL, suggesting that they were of conventional CTL origin. According to their fine specificity pattern, the CTL clones were divided into two major groups (A and B) which were further subdivided into five and three subgroups, respectively. The specificity of group A clones was essentially the same as that of the standard polyclonal CTL population except for a variable level of natural killer-like activity by some of the CTL clones. That is, group A clones did not efficiently lyse the insusceptible variant tumors nor any of Friend-Moloney-Rauscher-positive tumors tested, but they showed strong lytic activity to susceptible tumors and iododeoxyuridine-treated insusceptible variants. Thus, their CTL activity appeared to be strictly directed to Gross cell surface antigen-positive tumors that are susceptible to polyclonal anti-AKR/Gross virus CTL. In contrast, group B clones could lyse both susceptible and insusceptible variant tumors and also a Friend virus-induced tumor (FBL3). Therefore, as defined by these CTL clones, at least two distinct antigenic systems (A and B), each with several antigenic determinants, appeared to be present. Because recent findings suggested that most of the polyclonal anti-AKR/Gross virus CTL activity appeared to be directed to N-ecotropic proviral determinants, we further investigated the nature of these two antigenic systems by use of additional target cells including lipopolysaccharide (LPS)-stimulated spleen cell blasts from AKXL recombinant inbred strains and retrovirus-infected fibroblasts. Group A clones could lyse all LPS blasts derived from AKXL recombinant inbred strains containing the AKV-1 proviral genome, but lysed only very insufficiently or did not lyse AKV-1-negative blasts containing the AKV-3 and/or AKV-4 provirus.(ABSTRACT TRUNCATED AT 400 WORDS)