Natural thymocytotoxic autoantibodies (NTA) have been suggested to contribute to the T cell mediated immune dysfunction prevalent in NZB mice. However, direct studies to confirm these suggestions have been difficult to perform due to the absence of large amounts of pure NTA. To overcome this problem, we generated a naturally occurring monoclonal antibody from an NZB mouse which possesses the serological characteristics of serum-NTA. Herein, we report the in vitro functional characteristics of this monoclonal antibody designated SAG-3. In vitro treatment of spleen and lymph node cells from BALB/c mice with SAG-3 and complement eliminated most of the proliferative response to Con-A and moderately reduced the response of these cells to PHA-P and PWM. Complement mediated cytolysis of SAG-3 reactive cells significantly reduced the capacity of unprimed lymph node cells to generate primary CTL responses in vitro against allogeneic cells. However, only a partial decrease in CTL activity was observed when primed secondary CTL effector cells were treated, due primarily to the depletion of CTL's specific towards MHC class I antigens. Furthermore, a moderate reduction in antigen-specific helper T cell function was observed. Pretreatment of responder cells with SAG-3 + C' did not appear to reduce the proliferative MLR response to a wide range of stimulator cells. In addition, contrary to previous reports on serum-NTA, SAG-3 appears not to be reactive against Con-A induced suppressor T cells. Finally, by FACS analysis SAG-3+ thymocytes constitute an overlapping population of Lyt-2+ and L3T4+ thymocytes. These findings are discussed in comparison to previous reports on NTA.