The NOD mouse is a model of human juvenile type I diabetes mellitus. As in humans and in the BB rat model, the development of diabetes in NOD mice is accompanied by evident manifestations of cell-mediated and humoral autoimmunity. Beside autoantibodies directed at putative islet cell antigens, NOD sera contain antibodies with specificity for lymphocyte cell-surface determinants. Here we demonstrate that these anti-lymphocyte antibodies have the same characteristics of target cell specificity, of isotype, and of temperature reactivity, as do natural thymocytotoxic autoantibodies (NTA) from lupic NZB mice, or from mice undergoing polyclonal B cell activation. We also demonstrate that the thymocytotoxic activity of NOD sera is not due to cross-reactive anti-insulin antibodies. Biochemical characterization of the determinants recognized by these anti-lymphocyte antibodies reveals two membrane-associated proteins of 28 and 33 kD, partially similar to the two peptides recognized by NTA from NZB mice (30 and 33 kD). Altogether, these results suggest that NOD mice develop manifestations of polyclonal B cell activation similar to those observed in lupus-prone mice. The relationship of these anomalies with the organ-specific pancreatic disease remains to be properly evaluated.