To evaluate the effects of amylin and calcitonin-gene-related peptide (CGRP) as anti-insulin agents in hepatic tissue, we have studied whether these two agents counteracted the action of insulin on glycogen metabolism in isolated rat hepatocytes. In this system insulin stimulates [14C]glucose incorporation into glycogen and activates glycogen synthase. Incubation of the cells with insulin in the presence of amylin or CGRP markedly blocked the insulin stimulation of these two parameters, whereas amylin or CGRP acting alone did not induce any effect. We also examined the ability of amylin and CGRP to modify the anti-glucagon effects of insulin. In the presence of 100 nM-amylin or -CGRP, 10 nM-insulin was almost unable to counteract the inactivation of glycogen synthase and the activation of phosphorylase induced by glucagon. In contrast, neither amylin nor CGRP modified the effect of glucagon on these two enzymes. Our results indicate that amylin and CGRP are able to impair the action of insulin on hepatic glycogen metabolism.