We, as well as many other investigators, have been studying the regulation of immune responses to insulin as a model system of H-2 linked immune response (Ir) gene control. Although antibody responses by mice to heterologous insulins are qualitatively controlled, antibodies that are generated to one species of heterologous insulin cross react extensively with other species. The exquisite control of responsiveness is regulated by T cells that appear to recognize differences in the amino acid sequences of the A-chain loop of insulin. Our previous studies of the mechanism(s) by which Ir genes regulate T cell activity to insulin have been confined to an adoptive transfer model because traditional cell culture techniques using normal or insulin-primed spleen cells have failed to generate insulin-specific plaque-forming cell responses in vitro. In this communication we demonstrate that more vigorous immunization protocols and the use of lymph node T cells as a source of helper T cells can circumvent this problem. More importantly, all of the major features of the regulation of responses to insulin that have been observed in vivo are reflected in this in vitro system. Thus, these experiments provide the essential foundation for future dissection of the mechanism of Ir gene control of responses to insulin.