A series of experiments were performed to explore the role of complementing major histocompatability complex (MHC)-linked immune response Ir genes in the murine T cell proliferative response to the globular protein antigen pigeon cytochrome c. The functional equivalence of I-E-subregion-encoded, structurally homologous E(a) chains from different haplotypes bearing the serologic specificity Ia.7 was demonstrated by the complementation for high responsiveness to pigeon cytochrome c of F(1) hybrids between low responder B 10.A(4R) (I-A (k)) or B 10.S (I-A(8)) mice and four low responder E(a)- bearing haplotypes. Moreover, this Ir gene function correlated directly with both the ability of antigen-pulsed spleen cells from these same F(1) strains to stimulate pigeon cytochrome c-primed T cells from B10.A or B10.S(9R) mice, and with the cell surface expression of the two-chain Ia antigenic complex, A(e):E(a), bearing the conformational or combinatorial determinant recognized by the monoclonal anti-Ia antibody, Y-17. The B 10.PL strain (H-2(u)), which expresses an Ia.7-positive I-E- subregion-encoded E(a) chain, failed to complement with B10.A(4R) or B10.S mice in the response to pigeon cytochrome c. However, (B10.A(4R) x B10.PL)F(1) and (B10.S x B10.PL)F(1) mice do express A(k)(e):E(u)(a) and A(8)(e):E(u)(a) on their cell surface, although in reduced amounts relative to A(k,s)(e):E(k,d,p,r)(a) complexes found in corresponding F(1) strains. This quantitative difference in Ia antigen expression correlated with a difference in the ability to present pigeon cytochrome c to B 10.A and B 10.S(9R) long-term T cell lines. Thus, (B10.A(4R) x B10.PL)F(1) spleen cells required a 10-fold higher antigen dose to induce the same stimulation as (B10.A(4R) x B10.D2)F(1) spleen cells. In addition, the monoclonal antibody, Y-17, which reacts with A(e):E(a) molecules of several strains, had a greater inhibitory effect on the proliferative response to pigeon cytochrome c of B10.A T cells in the presence of (B10.A(4R) X B10.PL)F(1) spleen cells than in the presence of (B10.A(4R) X B10.D2)F(1) spleen cells. These functional data, in concert with the biochemical and serological data in the accompanying report, are consistent with the molecular model for Ir gene complementation in which appropriate two-chain Ia molecules function at the antigen-presenting cell (APC) surface as restriction elements. Moreover, they clearly demonstrate that the magnitude of the T cell proliferative response is a function of both the concentration of nominal antigen and of the amount of Ia antigen expressed on the APC. Finally, the direct correlation of a quantitative deficiency in cell surface expression of an Ia antigen with a corresponding relative defect in antigen-presenting function provides strong independent evidence that the I-region-encoded Ia antigens are the products of the MHC-linked Ir genes.