Human T peripheral blood lymphocytes were grown in continued culture using conditioned medium obtained from phytohaemagglutinin-stimulated, pooled human leucocytes. These cultured T cells (CTC) were tested in mixed lymphocyte culture (MLC) and cell-mediated lympholysis (CML) assays to determine the genetic specificity of their proliferative and cytotoxic responses. Primary responses were measured after initial in vitro stimulation by allogeneic cells, and secondary responses were measured after a second in vitro stimulation by allogeneic cells. Both primary and secondary proliferative responses were found to be stimulated by alloantigens controlled by the HLA region and, more specifically, by antigens of the HLA-D region, in accordance with the responses of normal peripheral blood T lymphocytes. When CTC were established from unsensitized PBL and then stimulated with allogeneic cells, they could respond by proliferation in MLC, but, in contrast to PBL, they did not show subsequent cytotoxic responses. On the other hand, CTC established from PBL that had been stimulated first with allogeneic cells in either primary or secondary MLC displayed high levels of cytotoxic reactivity in CML. The strongest cytotoxicity was directed against allospecificities controlled by the HLA region and specific for the MLC-stimulating cells, but lower levels of cross-reactive cytotoxicity were also observed.