Because surface Ig and Ia antigens cap independently, A.TH anti-A.TL serum combined with the indirect immunfluorescence technique could be used to test defined murine cell populations ofH-2 k haplotype for the presence of Ia antigens. Mitogen induced T- and B-cell derived blast cells, purified by velocity sedimentation at 1g, were tested for the expression of Iak antigens and then used both as stimulator cells and as target cells, in primary and secondary in vitro cytotoxic allograft responses. Fibroblasts, cortisone-resistant thymocytes, and nylon column purified splenic T cells were also included in these tests. Ia antigens were detected on 100% of LPS-induced blast cells, on 20%–30% of ConA-induced blast cells (100%Θ Thy-1 or antigen positive), but only to 5%–10% on PHA-blasts (100% Thy-1 antigen positive). Fibroblasts and nylon column purified splenic T cells were essentially Ia negative. Ia-positive allogeneic stimulator cells induced a far stronger in vitro cytotoxic T-cell response compared to Ia-negative stimulator cells; that is, there was a positive correlation between the expression of Ia antigens on the stimulator cells and the magnitude of cytotoxicity induced. This correlation was restricted to primary allograft responses. Ia antigens could not be detected as a target for killing in the cytotoxic effector phase, using both different target cells as well as the approach of “PHA dependent lysis” for detecting cytotoxic T lymphocytes.