Peripheral blood leukocytes from all normal humans tested have generated trinitrophenyl (TNP)-dependent cytotoxic activity after in vitro sensitization with TNP-modified autologous cells. Weak cytotoxic activity was generated during primary culture; total cytotoxic activity increased approximately 100-fold during secondary stimulation and another 5-fold during tertiary stimulation. The cytotoxic activity was shown to be TNP-dependent at both the sensitization and the lytic phases. Effectors lysed not only TNP-modified autologous cells but all TNP-modified human leukocyte targets tested, including those that shared no serologically defined HLA-A, -B, or -C locus determinants with the specific target. This cross-reactivity was species specific, since it was not observed when human effectors were tested on TNP-modified mouse targets. The TNP-dependent cytotoxic activity on autologous and unrelated cells was shown to be mediated by T cells without requirement for Fc-bearing cells; cytotoxic activity was recovered in subpopulations of cells positively selected by sheep erythrocyte-rosetting techniques and in subpopulations negatively selected by depletion of phagocytic cells, sIg-positive lymphocytes, and Fc-receptor positive lymphocytes. Furthermore, unlike ADCC mediated by anti-TNP antibody, the cytotoxic activity was inhibited by TNP-modified autologous cells, but not by TNP-lysine, or TNP-conjugated chicken red blood cells. We conclude that some human cytotoxic T cells raised against TNP-modified autologous cells because they express a receptor for TNP in conjunction with cell surface determinant(s) that are widely shared among humans but not shared with mice.