The metabolism of endogenous triacylglycerols (TG) was studied in perfused working hearts of control and 12-day-old streptozotocin-treated diabetic rats. TG synthesis was assessed by the incorporation of exogenous [9,10(-3)H]palmitate. TG lipolysis was assessed by the following two methods: 1) measurement of the decrease in [14C]TG after prior in vivo isotopic prelabeling with [1-14C]palmitate and 2) calculation of the change in TG content as TG synthesis minus TG lipolysis. Control and diabetic hearts were perfused with buffer containing substrate concentrations characteristic of the in vivo state [normal: 9 mM glucose, 0.5 mM free fatty acid (FFA); diabetic: 27 mM glucose, 1.2 mM FFA]. Diabetic hearts were also perfused under normal substrate conditions. In diabetic hearts perfused under diabetic conditions elevated TG levels were maintained, lipolysis was reduced or unchanged (depending on the method of determination), and synthesis was enhanced. Oxidation of TG fatty acids (TGFA) was not impaired. Control hearts showed net lipolysis coupled with lower rates of exogenous FFA uptake and TG synthesis. In diabetic hearts perfused under normal substrate conditions lipolysis and TGFA oxidation were markedly enhanced. Thus we suggest that the basis of TG accumulation seen in the diabetic heart is due to both inhibition of lipolysis and enhancement of synthesis resulting from high levels of exogenous FFA and glucose.