Regulation of the mobilization of endogenous fatty acids from lipid droplet triglyceride was investigated in isolated hepatocytes from normal-fed rats. Mobilized fatty acid was entirely accounted for in oxidation products (CO2 plus acid-soluble products). Dibutyryl cyclic AMP (DBcAMP) stimulated the mobilization of endogenous fatty acids by over 50% although no change in free fatty acid (FFA) levels was observed. In the presence of tetradecyglycidic acid (TDGA), a specific inhibitor of mitochondrial fatty acid oxidation, DBcAMP was unable to promote endogenous fatty acid oxidation; instead, the nucleotide increased the FFA level. This effect was blocked by the lysosomal inhibitor, chloroquine, supporting the concept that a lysosomal lipase is involved in the degradation of endogenous triglyceride. These findings suggest that cyclic AMP stimulates lysosomal lipolysis by a mechanism that is independent of changes in fatty acid oxidation. Epinephrine alone slightly suppressed triglyceride mobilization. Epinephrine plus doxazosin slightly increased lipid mobilization. Epinephrine plus propranolol suppressed endogenous fatty acid oxidation to a level significantly below that induced by epinephrine plus doxazosin. These results suggest that alpha 1-adrenoceptor activation suppresses hepatic triglyceride mobilization. The weak stimulatory effects of beta-adrenoceptor activation on hepatocyte triglyceride mobilization compared with the potent stimulation by DBcAMP is probably attributable to the relatively low level of beta-adrenoceptors in hepatocytes from the mature male rats used in these studies. Further characterization of a possible mutually antagonistic interaction between alpha 1- and beta 2-adrenoceptors in the regulation of hepatic triglyceride mobilization might be better accomplished in a species with hepatocytes that have a higher proportion of beta-adrenoceptors, e.g., guinea pig, rabbit, and dog.