Spleen cells obtained from hamsters bearing PARA-7 tumors greater than 1.0 cm were not reactive in microcytotoxicity assays unless preincubated overnight. The events occurring during in vitro incubation which lead to reversal of tumor-mediated suppression of cellular immunity were investigated. After 24 hr of incubation, supernatants overlying spleen cells from tumor-bearing hosts contained a factor which blocked cytotoxicity of simian virus 40 (SV40)3-sensitized spleen cells at the PARA-7 target cell level but not at the effector cell level. The preparations did not mediate antibody-dependent cellular cytotoxicity (ADCC). Opposite results were obtained in assays of culture medium overlying spleen cells from hosts with a tumor burden less than 0.1 cm. Although ADCC activity was present, no significant blocking was detectable. Treatment of inactive spleen cells with anti-hamster gamma-globlin in the presence of complement (anti-HGG + C) prevented activation and formation of blocking factor but did not impair the cytotoxic activity of already activated cells. Addition of SV40 antiserum to anti-HGG + C-treated cells led to effector cell activation, whereas heterologous virus-immune sera did not. Control studies established that the antibody-mediated recovery of cytotoxicity was not due to arming. Further studies showed that PARA-7 tumor antigen extract blocked at the effector cell level, not at the target cell level. Addition of PARA-7 extract to spleen cell supernatants mediating ADCC resulted in formation of a factor which blocked at the target cell level but not at the effector cell level. These data are compatible with the following interpretation. Spleen cell unresponsiveness is due to antigen blockade. Recovery of cytotoxicity occurs because antibody synthesized during the incubation period promotes elution of antigen from the effector cell surface. Thus, activation is accompanied by the generation of tumor antigen-antibody complexes.