Mouse spleen cells were fractionated and tested for their ability to stimulate a primary mixed leukocyte response (MLR). Several nonlymphoid populations were stimulatory, purified dendritic cells and 18-h adherent macrophages being the most effective. B and T lymphocytes and peritoneal washout cells were almost ineffective by comparison. This hierarchy of stimulator potential was observed whether the genetic difference between stimulator and responder was whole H-2 or H-2 subregion (K, D, I, I-J). Allostimulatory populations contained a high proportion of cells expressing I region determinants. The functional importance of these antigens was confirmed using specific anti-I region antisera. Thus, it appears that stimulator cells in the MLR are heterogeneous, Ia-bearing, and of "accessory" rather than lymphoid type.