Primary mixed glial cultures containing astrocytes, oligodendrocytes and macrophages have been cultured from cerebral hemispheres of neonate rats and examined by indirect immunofluorescence for the expression of class I and II RT1 major histocompatibility complex-coded antigens. None of these cells expressed detectable levels of either class I or II antigens except for the macrophages which were weakly class I positive. Treatment with lymphokine-containing supernatant from concanavalin A-activated splenic lymphocytes resulted in increased expression of class I antigens on all cells together with the appearance of class II RT1.B and RT1.D antigens on macrophages and a small proportion of type 1/protoplasmic astrocytes. The identity of the Ia antigens was confirmed by immunoprecipitation from lysates of surface-iodinated cells. The ability of lymphokine-treated mixed glial cultures to stimulate proliferation of allogeneic lymphocytes provides additional evidence of Ia induction. A possible role for these Ia+, putative antigen-presenting cells in delayed type hypersensitivity in the central nervous system is discussed.