In vitro studies were performed to determine the role of metabolic bioactivation in mediating immunosuppression by CCl4. Direct addition of CCl4 to naive spleen cell cultures sensitized with either sheep erythrocytes, DNP-Ficoll or lipopolysaccharide (LPS) resulted in a marked inhibition of the antibody forming cell (AFC) response to all three of the selected antigens at 3.0 mM concentration in culture. However, this inhibition was primarily due to the direct cytotoxic effects of CCl4 on spleen cells following 3-5 days of culture in the presence of the chemical as evidenced by a decrease in cell number and viability and by the absence of selective effects on T-cell dependent humoral responses which is contradictory to the effects observed in vivo. Co-incubation of splenocytes for 1 h with primary hepatocytes, but not with subcellular metabolic activation systems, such as S9 or microsomes, enhanced the immunotoxic effects of CCl4 in vitro. Interestingly, a 3-h co-incubation of spleen cells with metabolically active hepatocytes in primary culture resulted in an even greater potentiation of the immunotoxic effects of CCl4 as determined by the T-cell dependent IgM AFC response. Conversely, under identical conditions, CCl4 did not suppress humoral responses to the polyclonal B-cell activator LPS which is in agreement with the effects produced by in vivo exposure to CCl4. It is important to emphasize that for the metabolic activation studies (i.e. co-incubation with either S9, microsomes or hepatocytes), spleen cells were washed free of CCl4 immediately following the co-incubation period. Control splenocyte cultures (i.e. no metabolic activation system) incubated in the presence of CCl4 alone at 3.0 mM over a 3-h time-period, had no effect on spleen cell function, number or viability. In agreement with our previous findings which indicate that pretreatment of mice with inducers and inhibitors of the mixed function oxygenase system prior to CCl4 administration modulated the immunotoxic effects of CCl4 in vivo, these results lead us to conclude that immunotoxicity by CCl4 requires metabolic activation.