Exposure of 1321N1 human astrocytoma cells to fresh medium containing fetal bovine serum induced a marked increase in the subsequent ability of isoproterenol and forskolin to stimulate cAMP accumulation in intact cells, compared with cells exposed to fresh medium without serum. This "sensitization" of cAMP accumulation by serum was dose dependent, occurred rapidly, was maintained in the continuing presence of serum, and reversed rapidly upon removal of serum. Preliminary characterization of the sensitizing factor(s) in serum has been performed, but the factor(s) remain to be identified. Sensitization appeared to result from an increase in maximal response and not from changes in the potency of isoproterenol or forskolin. The protein kinase C inhibitor staurosporine inhibited serum-induced sensitization. Furthermore, down-regulation of protein kinase C almost completely eliminated the subsequent ability of serum to induce sensitization, indicating involvement of protein kinase C in the serum effect. Pretreatment of cells with pertussis toxin also markedly reduced subsequent sensitization induced by serum, suggesting involvement of a pertussis toxin-sensitive guanine nucleotide-binding protein in the pathway for serum-induced sensitization. The rate of cAMP degradation was not changed in sensitized cells, but some increase in adenylyl cyclase activity was retained in broken cell preparations from sensitized cells, suggesting increased synthesis of cAMP by adenylyl cyclase as the mechanism for sensitization.