Spleen cells from W/Fu rats 40 days or more after immunization with a syngeneic Gross virus-induced leukemia were unreactive in direct cytotoxic assays. Incubation of these immune cells at 37 degrees C for 12 hr or longer, in the absence of antigen, resulted in the appearance of specific cytotoxic reactivity. Other lymphoid cells from the immune rats also were activated upon in vitro incubation, but to a lesser extent. Experiments were performed to define the necessary conditions and the mechanism for the in vitro incubation. Activation was temperature dependent, occurring at 37 degrees C but not at 4 degrees C. Immune serum suppressed the activation, but normal rat serum also had some inhibitory activity. Passage of immune cells through a nylon column, before preincubation, prevented activation. In contrast, exposure to nylon after preincubation did not remove cytotoxic reactivity. These findings demonstrate the reversal of a central inhibition of immune cell activity. The explanations offered for this phenomenon included change in surface characteristics of the immune cells during in vitro incubation, and the possible need for an adherent helper cell.