The hormonal responsiveness of freshly isolated rat hepatocytes was compared to that of a) cold-preserved isolated hepatocytes and b) hepatocytes isolated from cold-preserved whole liver. Cold-preserved hepatocytes and cells isolated from cold-preserved whole liver increased phosphorylase alpha activity in response to norepinephrine (plus propranolol), vasopressin, angiotensin II and glucagon. However, the maximal response to these agents was smaller than that of freshly isolated hepatocytes. Basal phosphorylase alpha activity was increased in cold-preserved hepatocytes. Similarly, cold preservation decreased the accumulation of cyclic AMP induced by glucagon and the effects of norepinephrine (plus propranolol), vasopressin and angiotensin II on the production of inositol phosphates. Basal levels of cyclic AMP were similar in the three conditions studied but basal production of [3H]IP2 plus [3H]IP3 was increased in cold-preserved hepatocytes. There was a very small effect of beta-adrenergic activation on phosphorylase activity and a small accumulation of cyclic AMP in response to isoproterenol in the conditions studied.