Transforming growth factor β (TGF-β) is a potent immunosuppressive cytokine that is produced by neoplastic and normal cells. It has not been demonstrated directly, however, that TGF-β can inhibit antigen-specific T-cell responses to tumor cells in vitro. We show here that generation of antitumor cytotoxic T-lymphocyte (CTL) activity in mixed-lymphocyte tumor cultures of splenocytes from DBA/2 mice immunized with the syngeneic P815 mastocytoma + Corynebacterium parvum was consistently and profoundly inhibited when 0.675 to 10 ng/ml of TGF-β were added on Day 0 of culture. TGF-β added on Day 1 or later had little or no effect. In contrast to the results with P815 immune mice, mixed-lymphocyte tumor cultures established with splenocytes from P815 tumor-bearing hosts showed variable degrees of inhibition by TGF-β, depending on the stage of the ongoing in vivo immune response. Addition of recombinant murine tumor necrosis factor α (1,000 or 10,000 units/ml) partially reversed inhibition of CTL responses by TGF-β, while recombinant interleukin 2 nearly completely reversed the suppression. These data indicate that one level at which TGF-β may act to inhibit mixed-lymphocyte tumor cultures is that of cytokine production. To determine whether TGF-β also has any direct effect on CTL, P815-specific CTL clones derived from tumor-bearing host mice were utilized. We found that proliferation of rested CTL clones in response to tumor cells + interleukin 2 was inhibited by 5 ng/ml of TGF-β, while the interleukin 2-dependent reactivation of cytolytic activity was not affected by TGF-β. In contrast to rested CTL, when TGF-β was added to cultures of previously activated CTL, proliferation was not inhibited. These data demonstrate that TGF-β has profound inhibitory effects on the in vitro generation of effector CTL from tumor-specific murine splenocytes, and this inhibition may be an indirect result of suppressed cytokine production as well as a direct antiproliferative effect on CTL.