The response to heterologous insulin in H-2b mice is restricted to the A chain loop determinant(s) of beef insulin. The recognition of this specificity requires the expression of the immune response (Ir) gene epitope Ia.W39 which is absent from the I-Ab mutant B6.C-H-2bm12 (bm12) mice. This restriction could reflect the inability of H-2b antigen-presenting cells (APC) to present other insulin determinants or may reflect "self-major histocompatibility complex"-dependent influences on the generation of the T cell repertoire. To assess these possibilities we analyzed the genetic control and fine specificity of the insulin-specific T cell repertoire of H-2b mice by fusing the AKR thymoma BW5147 with T cells of C57BL/6 mice which had been immunized in vivo and challenged in vitro with beef insulin. The cloned hybridomas that we have produced respond to APC either alone or in conjunction with insulin by the production of interleukin 2. The insulin-specific hybridomas vary in their fine specificity such that some clones recognize a determinant(s) shared by beef, sheep and pork insulin and the isolated B chain, while other clones recognize a determinant(s) shared by beef and sheep insulin only, likely to involve amino acids 8 and/or 10 of the A chain loop. The presentation of insulin to these hybridomas is restricted by I-Ab, but not by Ia.W39. This analysis revealed that the insulin-specific immune potential in H-2b mice is of greater scope than previously defined and led us to consider, whether insulin nonresponder bm12 mice also possess a latent insulin-specific immune potential. Our study of the insulin-specific immune recognition by bm12 mice shows that these nonresponders do possess insulin-specific T cell clones. Despite the fact that the I-Ab and I-Abm12 gene products differ only by 3 amino acids, insulin-specific C57BL/6 and bm12 hybridomas are restricted to recognize exogenous antigen only in the context of C57BL/6 and bm12 APC, respectively. Furthermore, upon direct analysis of autoreactive subclones, a similar although not complete, restriction was observed. The implications of these findings for understanding the mechanism of Ir gene control are discussed.