The addition of lactate (5.6 mM) to a perfusion medium containing glucose (11 mM) stimulated the turnover of the cardiac triacylglycerol pool throughout the perfusion period as indicated by increased glycerol release in association with maintained levels of triacylglycerols. Attenuation of feedback inhibition of triacylglycerol lipase by fatty acids as a possible cause of the elevated triacylglycerol turnover rate should be ruled out, since tissue fatty acid levels were 3-times higher in glucose plus lactate perfused hearts than in hearts perfused with glucose as the sole substrate. The present findings are in favor of the notion that lactate enhances triacylglycerol turnover through increased glycerol 3-phosphate levels.