The ability of glucagon or epinephrine to increase cAMP levels in primary monolayer cultures of adult rat liver parenchymal cells was markedly reduced to pretreatment of the cells with the same hormone. Induction of cAMP production by glucagon (2 X 10(-7) M) was reduced to a plateau level of about 30% of that in untreated cultures by 1 h of glucagon (2 X 10(-7) M) pretreatment, and induction by epinephrine (10(-5) M) pretreatment. The time courses of the stimulation of cAMP production by each hormone were similar in control and hormone-pretreated cells. There was no change in the concentration of hormone producing half-maximal stimulation of cAMP accumulation by epinephrine in desensitized cells and only a small increase in the concentration of glucagon producing half-maximal cAMP induction. Desensitization by glucagon was reversed to about 70% of control levels 1 h after placing treated cells in hormone-free medium, whereas desensitization by epinephrine was poorly reversible. Neither desensitization nor its reversal was affected by inhibition of protein or RNA synthesis. Cells pretreated with glucagon were desensitized equally toward glucagon and epinephrine, whereas epinephrine pretreatment caused, predominantly, desensitization toward epinephrine. Pretreatment of cultures with dibutyryl cAMP resulted in less inhibition of the stimulation of cAMP production by epinephrine than did pretreatment with the hormone itself. Depending on the cell preparation used, dibutyryl cAMP was either as effective or less effective than glucagon itself, but always caused some desensitization. The data are consistent with there being hormone-specific and nonspecific components involved in the overall process of desensitization toward glucagon and epinephrine, with a nonspecific component mediated by cAMP.