Naturally occurring thymocytotoxic autoantibodies (NTA) have been suggested to be the cause of thymic atrophy and T cell disorders in human and murine lupus. Definitive studies on NTA's role in the induction of SLE, however, have been lacking due to the lack of a pure source of NTA. Although it is clear that NTA are a heterogeneous group of antibodies, the nature of their antigens has remained obscure. We report the characteristics of a monoclonal NTA, designated SAG-3, which appears more reflective of the activities previously reported of serum NTA than other NTA-secreting clones. SAG-3 is an IgM autoantibody cytotoxic for 80 to 90% of thymocytes, 20 to 25% of splenic lymphocytes, 25 to 30% of lymph node cells, and less than 3% cortisol-resistant thymocytes, bone marrow, and fetal liver cells. SAG-3 is murine-specific without reactivity towards rat, hamster, or guinea pig, and appears very early in thymic development, on day 17 fetal thymocytes. SAG-3 is equally cytotoxic against several strains of mice, including both Thy-1.1 and Thy-1.2 allotypes, and the cytotoxicity is absorbed by brain but not liver cells. Reactive thymocytes occurred throughout the cortical regions of the thymus, indicating preferential affinity towards immature thymocytes. Although the serologic activities of SAG-3 suggest that Thy-1 alloantigen is its target, SAG-3 antigen is found to be distinct from Thy-1 and also from Lyt-1, Lyt-2, or L3T4 antigens. The binding of SAG-3 to thymocytes could be competitively inhibited by NTA-positive NZB sera.