The present study has addressed the involvement of Ia determinants in the in vitro generation of antigen-specific cytotoxic T lymphocyte (CTL) responses. It demonstrated that the in vitro generation of TNP-specific CTL responses strictly requires responder T cell recognition of self-Ia determinants expressed by accessory cells, and that this recognition could be specifically inhibited by monoclonal anti-Ia-antibodies. The generation of TNP-specific CTL responses was unaffected by the presence of anti-I-A antibodies or the absence of accessory cells when cultures were performed in the presence of an exogenous source of T helper cell factors, Con A SN. Thus, these results indicate that T helper cell recognition of self-Ia determinants expressed by accessory cells is required for the generation of TNP-specific CTL responses. These results preclude the possibility that accessory cells perform only immunologically nonspecific roles in the generation of hapten-specific CTL, but instead demonstrate that accessory cells function in such responses as Ia-bearing antigen-presenting cells for the activation of self-Ia-specific T cells.