An immunoregulatory role has recently been attributed to the discrete subset of major histocompatibility complex class I-restricted NK1+ mature heat-stable antigen- (HSA-) thymocytes expressing an unusual Vbeta8-biased T cell receptor repertoire. NK1+ T cells are the main interleukin (IL)-4 producers upon priming. We have studied the size and the function of this subset in the nonobese diabetic (NOD) mouse, a model of spontaneous T cell-mediated autoimmune insulin-dependent diabetes. This study was complicated by the absence in this strain of the NK1.1 allele, the only one for which an antibody is available. To circumvent this difficulty, the cells, hereafter designated the NK1+-like T subset, were characterized by the use of monoclonal antibodies which showed the Vbeta8 bias in the CD44+ Ly-49+ MEL-14- 3G11- thymocyte subset of non-autoimmune strains and of its absence in class I-deficient (beta2-microglobulin-/-) mice. A clear deficit in the number of NK1+-like cells was evidenced at 3 weeks of age in NOD mice. It was still present at 8 weeks of age in the double-negative CD4-CD8- population. The functional anomaly was even more striking: NOD mouse NK1+-like thymocytes virtually lacked the ability to produce IL-4 at 3 weeks and still showed a very reduced capacity at 8 weeks. NK1+ T cell deficiency was also suggested in the periphery by the reduction of Ly-49A+ cells in the spleen of 3- and 8-week-old NOD mice and the absence of short-term production of IL-4 in vitro by NOD mouse spleen cells 90 min after the administration of anti-CD3 antibody, a response attributed to NK1+ T cells. Taken together, these data demonstrate a very early defect in NK1+-like T cells which could be involved in the genesis of autoimmunity in NOD mice through a deficiency in Th2 cell function.