During treatment of primary cultured hepatocytes with dexamethasone for several hours, cyclic AMP formation and glycolysis by glucagon increased dose dependently. In the cells pretreated with dexamethasone, the output of intracellular cyclic AMP increased significantly (p less than 0.01) with glucagon of 2.8 X 10(-9) M or more, and the amount of glucose released also increased significantly (p less than 0.01) with glucagon of 2.8 X 10(-8) M or more, compared to the control cells. Moreover, treatment with dexamethasone increased the stimulatory effect of guanosine-5'-triphosphate (GTP), guanyl-5'-yl-imidodiphosphate on adenylate cyclase and significantly increased the stimulatory effect of fluoride. In the rat hepatocytes primarily cultured with dexamethasone for several hours, the stimulatory effect of GTP and fluoride on adenylate cyclase increased time dependently. These data indicate that the glucocorticoid regulates the sensitivity of adenylate cyclase at distinct loci of the postreceptor system.