The expression of Ia antigens by accessory cells participating in in vitro primary antibody responses, including responses under Ir gene control, has been investigated. The ability of radiationresistant, non-T, non-B, spleen-adherent cells to function as accessory cells has been demonstrated both for the Ir gene-controlled response to TNP-(T,G)-A--L and for the response to TNP-KLH, for which no Ir gene control has been demonstrated. Pretreatment of these adherent accessory cells with anti-Ia reagents and complement totally abrogated the accessory function of this population for responses to both antigens. This abrogation of accessory function resulted from the elimination of accessory cells, rather than from any active suppression of responsiveness, either by carry-over of anti-Ia antibody or antigen-antibody complexes. By using appropriate recombinant strains and specific anti-Ia reagents, it has further been demonstrated that Ia determinants encoded in at least two I subregions, I-A and I-E/C, are expressed on accessory cells for responses to TNP-KLH and TNP-('T,G)-A--L; and that determinants encoded in at least two I subregions are expressed simultaneously on the same cells. The data presented also suggest that the functionally active Ia-positive accessory cell is itself either nonphagocytic or constitutes a subpopulation of phagocytes expressing Ia antigens encoded in at least two I subregions.