A monoclonal natural thymocytotoxic autoantibody NTA204 established from an autoimmune-prone NZB mouse reacted with the majority of thymocytes, all peripheral B cells, granulocytes and bone marrow myeloid cells, but not with peripheral resting T cells of normal mice. In aged NZB/W F1 mice with overt autoimmune disease, the population of NTA204+ CD4+ CD25- T cells was remarkably increased. The NTA204 antigen could be induced on splenic T cells from normal healthy mice as early as 3 hr after the initiation of culture with stimulant Con A, and was expressed on the vast majority in the 48-hr culture. The expression preceded that of other T cell activation antigens tested, CD25 and CD45R. Cell cycle analysis suggested that NTA204 is expressed at an early phase of G1A. T cells, particularly CD8+ T cells, in the allogeneic mixed lymphocyte culture (MLC) could be divided into two populations, NTA204+ and NTA204-. By immunohistochemical analysis, 30% of NTA204+ CD8+, but few NT204- CD8+ T cells were intensely positive for large cytoplasmic granules of perforin, an important cytolytic mediator of cytotoxic T cells. Thus the increased population of NTA204+ T cells in aged NZB/W F1 mice appear to be activated T cells and might be at least partly involved in the pathogenesis of disease in these mice. Immunoblotting analysis of Con A-activated splenic T cells showed that NTA204 molecules have a molecular mass of 49 Kd.