Vasopressin stimulates several metabolic processes, including glycogenolysis, gluconeogenesis, and fatty acid oxidation, and promotes lipolysis in rabbit and hamster suprarenal adipose tissue. This study was conducted to determine whether arginine vasopressin (AVP) stimulates the metabolism of triacylglycerols in the isolated perfused rabbit heart. Since the basal output of glycerol in the rabbit heart is very low, the triacylglycerol pool was labeled with [3H]triolein, and triacylglycerol metabolism was followed by analysis of the radioactive products in the perfusate. Administration of AVP (100 ng, 92 pmol) produced a 10-fold increase in the perfusate radioactivity associated with free fatty acids and mono- and diglycerides, as well as an 8-fold increase in the effluent radioactivity associated with triacylglycerol. The V1-receptor antagonist d[(CH2)5Tyr(Me)]AVP blocked the AVP-induced increase in the output of radioactivity in a dose-related manner. The V2-receptor agonist desmopressin (DDAVP) did not increase the outflow of radioactivity. Likewise, AVP-induced release of radioactivity was inhibited when Ca2+ was omitted from the perfusion buffer. Analysis of total lipid extracts of hearts labeled with [3H]triolein showed that the residual radioactivity was associated almost exclusively with authentic triolein both before and after AVP treatment. These data suggest that AVP promotes triacylglycerol mobilization and utilization and that these processes are Ca2+ dependent and mediated by the V1-receptor. Since free fatty acids derived from triacylglycerols are the preferred metabolic substrate for the heart and since plasma AVP levels increase in cardiac stress states such as shock, these findings suggest a metabolic function of AVP in cardiovascular stress states.