Treatment of intact hepatocytes with cholera toxin at 37 degrees C caused a stable activation of adenylate cyclase activity after a lag period of around 10 min. The presence of either insulin (10 nM) or glucagon (10 nM) in the incubation medium had little effect on this lag period; however, these hormones markedly attenuated the maximal activation of adenylate cyclase activity that could be achieved by treatment with cholera toxin. Such actions of insulin and glucagon were dose-dependent, with EC50 values (concn. giving 50% inhibition) of 0.20 nM for insulin and 0.49 nM for glucagon, and were not additive. Treatment of intact hepatocytes with either glucagon or insulin did not affect the ability of cholera toxin to cause the ADP-ribosylation of the 45 kDa alpha-subunit of the stimulatory guanine nucleotide regulatory protein, Gs, in intact hepatocytes. It is suggested that treatment of intact hepatocytes with either insulin or glucagon attenuates the stimulatory action of ADP-ribosylated Gs on adenylate cyclase.