Diabetes in rats was induced with streptozotocin (100 mg/kg); myocardial cells (myocytes) were isolated from the hearts 3-4 days later. Diabetic myocytes were characterized as having the same viability and ATP content as control myocytes, but the yield was reduced. The triacylglycerol content of diabetic myocytes was elevated by 3.7-fold; this resulted in an increased rate of glycerol output during subsequent incubations. There was a stoichiometric relationship between the decline in the cellular triacylglycerol content and the release of glycerol into the incubation medium. Isoproterenol stimulated the output of glycerol from control myocytes by about twofold, but the stimulation of glycerol output from diabetic myocytes by isoproterenol was markedly less. The combination of 1-methyl-3-isobutylxanthine with isoproterenol or 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate also failed to produce the same lipolytic response in diabetic myocytes as in control myocytes. Triacylglycerol-loaded myocytes from control rats, prepared by including palmitate in the isolation buffers, were also characterized as having increased basal rates of glycerol output and a reduced lipolytic response to isoproterenol. The level of free fatty acids in diabetic myocytes was 2.8-fold greater than in myocytes from control hearts. The intracellular accumulation of free fatty acids in these quiescent populations of diabetic myocytes may limit the ability of catecholamines to produce a further stimulation of lipolysis.