The murine T cell response to heterologous insulins provides a good model system for studying the mechanism of immune response (Ir)-gene function, since insulin is a small, chemically well-defined molecule. H-2b mice respond predominantly to A chain loop determinants of beef insulin, presented by the I-A epitope Ia. W39. However, using a library of insulin-specific T cell hybridomas (THy), we previously found that immunization of H-2b mice with beef insulin activates a much wider population of T cells than are detected in T cell proliferation assays. Using such cloned THy we were able to study Ir-gene control at the level of antigen presentation. We compared the ability of the various THy to induce differentiation in I-A-matched B cells in response to antigen. Although both A and B chain-reactive clones respond with interleukin 2 production, they differ markedly in their potential to activate B cells in that only the former are able to induce B cell differentiation in the presence of the intact beef insulin molecule. The latter, however, can serve as helper cells in the presence of isolated B chain, and can synergize with a suboptimal concentration of A chain-reactive THy to induce an optimal B cell response. These results suggest that the insulin molecule is presented by I-Ab antigen-presenting cells in a very specific configuration that allows more effective T cell recognition of the A chain loop than the B chain determinants. To explain the discrepancy between the interleukin 2 assay and the induction of polyclonal activation, it can be assumed that in the former assay antigen is presented by macrophages, while presentation by B cells is necessary for induction of polyclonal activation. Macrophages are able to process the intact beef insulin molecule and, therefore, present B chain determinants, while nonimmune B cells may be unable to process antigen and could present B chain determinants only when the isolated B chain is given as antigen.