The human autologous pair of a cytotoxic T cell clone and tumor line could be very useful for the investigation of the specific destruction of autologous tumor cells by cytotoxic T lymphocytes (CTL), including analysis for tumor-specific antigen possibly of the rejection type, clonotypic T cell antigen receptors and biochemical characteristics of the cytotoxic molecules produced from CTL. We established such a pair of autologous specific CTL clone TcHMC-1 and target clone HMC-1-8 that were derived from the metastatic pleural effusion of a patient with mammary carcinoma. TcHMC-1 showed more than 60% specific cytotoxicity against HMC-1-8, and it was confirmed, using cold target inhibition assays, that this clone did not demonstrate nonspecific cytotoxicity against a allogeneic targets as well as the natural killer (NK) cell activity. We also confirmed the in vivo action of TcHMC-1 against HMC-1-8 cells by the Winn assay in nude mice. Using anti-CD3, 4, and 8 as well as anti-class I monoclonal antibodies (mAbs), it was demonstrated 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 (MHC) class I products. We developed mAb 3A2 reacting with the specific antigens on HMC-1-8 that could play an important role in this autologous pair. This mAb inhibited selectively the cytotoxic action of TcHMC-1 against HMC-1-8, and identified a molecule with approximately 92 kd m.w. 3A2-defined antigen was highly expressed on autologous primary breast carcinoma tissue, but not on normal mammary gland in the same patient. Moreover, this antigen can be detected on approximately 50% of human allogeneic breast carcinomas, but not on other neoplastic cells except for 1 out of 10 prostatic carcinomas. It was also suggested that 3A2-defined antigen is not murine mammary tumor virus-related antigen.