Genetic control of the T lymphocyte proliferative response to staphylococcal nuclease was investigated with the sensitive peritoneal exudate, T lymphocyte enriched cells (PETLES) assay. All mouse strains examined responded to nuclease when immunized with large doses (0.6 nmole) of the antigen in complete Freund's adjuvant. Nevertheless, control of responsiveness by major histocompatibility (MHC)-linked immune response (Ir) genes was discerned when the nuclease responses were normalized to the PPD response of the same strain and compared among strains possessing the same non-H-2 background. In addition, lowering the immunization dose enhanced the difference between low and high responder strains. Genetic mapping studies carried out under these limiting conditions confirmed the earlier findings of an Ir-Nase gene mapping to the I-B subregion of the H-2 complex on the basis of low responsiveness by the B10.A(4R) and B10.A(5R) recombinant strains. However, immunization of these recombinant strains with 0.6 nmole of nuclease demonstrated a difference in their responsiveness, suggesting that the genetic control might be more complex. This suggestion was confirmed by cross-stimulation studies of nuclease-primed PETLES with fragments of nuclease. The pattern of the proliferative responses was found to be controlled by MHC-linked genes mapping in the I (and possibly the K and D) region, and appeared to be unique for each haplotype. In the case of the 4R and 5R recombinants the patterns appeared different from either that for the high responder B10.A or that for the low responder B10 parental strains. These results suggest that several Ir genes are involved in the T cell proliferative response to nuclease. Furthermore, the fact that fragments of nuclease, which possess little of the tertiary structure of the native molecule, could stimulate a proliferative response in PETLES immunized to whole nuclease suggests that T cells, in contrast to most antibodies, recognize antigenic determinants that possess a similar conformation in both the native molecule and the fragments.