Hapten-conjugated cells of the dendritic epidermal T cell (DETC) line AU16 induce specific immunologic tolerance in vivo and inhibit the proliferation of native T cells in response to hapten-bearing dendritic cells in vitro. The purpose of this study was to test the hypothesis that these activities of DETC are mediated by a cytotoxic mechanism. In addition, because AU16 cells, unlike most DETC, express a TCR-alpha beta, we compared their activity in the in vivo and in vitro assays with a TCR(+)-gamma delta DETC line (T245) and an TCR(+)-alpha beta, FITC-specific T cell line (T4/28). Hapten-conjugated AU16 and T245 cells, but not T4/28 cells, induced tolerance to FITC in vivo. In addition, the proliferative response of naive T lymphocytes to gamma-irradiated, hapten-bearing APCs was inhibited by the addition of gamma-irradiated, FITC-conjugated DETC to the cultures on day 0. To determine whether the T cell or the APC was the target, AU16 cells were added to the cultures on day 4, when few APCs remained. FITC-conjugated AU16 and T245 cells profoundly inhibited [3H]thymidine incorporation by the T cells, indicating that the T cell was the target of the DETC. Furthermore, AU16 cells reduced the number of T cells in the in vitro proliferation assay, suggesting that inhibition of [3H]thymidine incorporation was a result of the cytotoxic activity of DETC. We speculate that cytotoxicity may also account for the ability of DETC to induce tolerance in vivo.