Short-term and long-term biological activities were studied in adult rat hepatocytes cultured in the presence of the insulin analogues des-(B26-B30)-insulinamide, [Tyr~25]des-(B26-B30)-insulinamide and [HisB25]des-(B26-B30)-insulinamide. When compared to insulin, full potency of des-(B26-B30)-insulinamide has been reported in rat adipocytes and an enhanced potency has been reported for the other analogues. Steady state binding characteristics of the analogues to hcpatocytes were indistinguishable from those of native insulin with half-maximal binding occurring at concentrations of about 0.8 nmol/I. Half-maximal effects for the stimulation of glycolysis and inhibition of basal and glucagon-activated glycogenolysis required identical concentrations for insulin and all 3 analogues. Induction of the key glycolytic enzymes glu-cokinase and pyruvate kinase as well as the inhibition of glucagon-dependent induction of phosphenolpyruvate carboxykinase also required identical concentrations of insulin and the 3 analogues. These data confirm that in cultured hepatocytes the C-terminal amidation of des-(B26-B30)-insulin results in a molecule with full in vitro potency. In contrast to data obtained in adipocytes, the des-(B26-B30)-insulin-amidated analogues with tyrosine or histidine substitutions at position B25 are equally as potent as native insulin in eliciting biological responses in rat hepatocyte culture.