A possible requirement for class II major histocompatibility complex (Ia) molecules in the initial activation of cytotoxic T lymphocyte precursors (CTLp) for allocytotoxic responses was investigated. To avoid possible interaction with other alloreactive cell types, a highly purified population of Lyt-2+ splenocytes was used as a source of CTLp. In the light of preliminary results indicating that Lyt-2+ CTLp, even in the presence of interleukin 2 (IL2), could best be triggered into mature CTL in vitro by cells known to be Ia+, we examined whether an interaction of CTLp with Ia antigens (either on syngeneic accessory cells or on allogeneic stimulators) played a role in the development of allocytotoxicity. Results from experiments done with C57BL/6 Lyt-2+ splenocytes co-cultured with P815 stimulator cells and IL 2 showed that the early activation of CTLp was independent of Ia+ syngeneic accessory cells: (a) flow microfluorometry analysis of the responder population at the beginning or after 1 or 3 days of co-culture did not reveal the presence of Ia+ cells; (b) procedures for removal of residual Ia+ cells or of dendritic cells from the responder population before co-culture did not affect the development of cytotoxicity; (c) co-culture with monoclonal antibodies against syngeneic Iab antigens did not inhibit the CTLp activation. By comparing an Ia+ P815 tumor line with its Ia- clone as allogeneic stimulator cells, it was found that the CTLp activation was also independent of Ia alloantigen on the stimulator cells. The response against both the Ia+ and the Ia- stimulator cell types was not inhibited by monoclonal anti-L3T4 present in the co-culture, indicating that these responses were not affected by residual L3T4 helper cells.