We have recently demonstrated that the superantigen staphylococcal enterotoxin A (SEA) targets in vitro activated cytotoxic T lymphocytes against tumor cells expressing major histocompatibility complex (MHC) class II antigens. In this report we analyze the use of SEA as an immunoactivator in vivo. Treatment of mice with SEA activated a fraction of CD3+ T cells apparently as a function of their T cell receptor V beta expression. SEA induced interleukin-2 receptor expression and proliferation in both CD4+ and CD8+ T cells. This proliferative response was dose-dependent (0.1-100 micrograms/mouse), peaked during day 1 after treatment and declined to background levels within 4 days. The cytotoxic response, measured as cytotoxicity to SEA-coated MHC class II+ target cells (staphylococcal-enterotoxin-dependent cell-mediated cytotoxicity, SDCC), was maximal at a dosage of 1 microgram SEA/mouse. The SDCC was confined to the CD8+ T cell compartment, peaked 2 days after treatment and declined to background levels within 4 days. A second injection of SEA on day 5 after the first SEA treatment resulted in SDCC function with kinetics and magnitude identical to that seen after one injection. These results pave the way for the use of SEA in the treatment of MHC class II+ tumors.