In the present studies, cAMP, a secondary messenger historically viewed as a negative mediator of immune responses, was demonstrated to possess immunoenhancing activity at low concentrations. In parallel experiments the adenylate cyclase inhibitor, 2',3'-dideoxyadenosine, produced a marked inhibition of humoral and proliferative immune responses suggesting that cAMP at physiologically relevant concentrations acts as a critical second messenger in immune responses. Direct addition of dibutyryl cAMP (10-100 microM), a membrane permeable cAMP analog, to mouse spleen cell cultures produced a marked and dose-related increase (25-100%) in humoral immune responses as measured by the primary IgM antibody forming cell response to the antigen, sheep erythrocytes. Over a similar concentration range, dibutyryl cAMP (5-50 microM) also dose-dependently enhanced (25-50%) phorbol 12-myristate 13-acetate/ionomycin-stimulated lymphoproliferation. Incubation of spleen cells with 2',3'-dideoxyadenosine (40-80 microM), an adenylate cyclase inhibitor, for 30 min depressed significantly the basal level of intracellular cAMP. 2',3'-dideoxyadenosine treatment also significantly decreased both the antibody forming cell response and the proliferative response in a dose-dependent manner. Interestingly, the antibody forming cell response exhibited significantly greater sensitivity to inhibition by 2',3'-dideoxyadenosine than the lymphoproliferative responses. The critical role for cAMP as a positive immunoregulatory signal is further supported by the fact that the immunosuppression by 2',3'-dideoxyadenosine could be reversed completely in the antibody forming cell response to sheep erythrocytes through the addition of dibutyryl cAMP into the culture medium. Partial but not complete reversal of the inhibitory effects of 2',3'-dideoxyadenosine on lymphoproliferation was also demonstrated by dibutyryl cAMP. Taken together, these results suggest that cAMP acts as a positive regulatory signal for immune responses as indicated by the fact that depletion of intracellular cAMP induces a marked inhibition of humoral and proliferative responses.