We demonstrated previously the establishment of a human cytotoxic T-cell clone, TcHMC-1, under culturing with recombinant interleukin that showed the specific cytotoxicity against an autologous breast tumor cell line, HMC-1-8. In the present study, the autologous tumor specific antigens that could be involved in this cytotoxicity were extracted by using n-butyl alcohol and were analyzed for their biochemical profiles. The cytotoxicity of TcHMC-1 against HMC-1-8 was inhibited by adding OKT3 and OKT8 monoclonal antibodies into the cultures, or by pre-sensitizing HMC-1-8 target cells by anti-major histocompatibility complex class I monoclonal antibodies. This suggests that T-cell antigen receptor molecule complexes Ti/T3 on TcHMC-1 and corresponding specific tumor antigens on HMC-1-8 are involved in the cytotoxicity under the restriction of major histocompatibility complex class I products. Precultures of TcHMC-1 with crude n-butyl alcohol extracts from HMC-1-8 cells enhanced the cytotoxic potentials of this clone as seen as mixed lymphocyte tumor cell cultures. This enhancement was dependent on dosage of crude n-butyl alcohol extracts and these TcHMC-1 cells were still cytotoxic specifically for HMC-1-8 targets, but not for other allogenic tumor lines including K562. However, HMC-1-8 crude n-butyl alcohol extracts could not enhance DNA synthesis of TcHMC-1 as assessed by incorporation of [3H]thymidine in the cells. Biochemical purification studies demonstrated that the HMC-1-8 tumor specific antigens were eluted into fractions containing molecules with molecular weights of approximately 200,000 on Sephadex G-200 column chromatography. The antigens were further separated into the fraction that was eluted with 0.4-0.5 M NaCl in an ionic strength on Mono Q fast protein liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of this fraction demonstrated three molecules with molecular weights of 26,000, 30,000, and 32,000 under reduced molecular conditions. The data suggest that these molecules could be tumor specific antigens that are involved in the cytotoxicity of cytotoxic T-cells against a human autologous tumor.