This paper shows that the palmitoyl-CoA hydrolase activity of postheparin serum of the rat is mainly derived from the liver. The identity of this activity with the heparin-releasable hepatic triacylglycerol hydrolase activity is established. The consequences of the different substrate specificities of the hepatic and extrahepatic enzymes for the measurement of the overall postheparin serum lipase activity are discussed. Treatment of the rats with either a corticosteroid or with streptozotocin was found to lower the lipolytic activity from the liver and to enhance the extrahepatic activity. Also in human postheparin serum, palmitoyl-CoA hydrolase activity is shown to behave identical with hepatic triacylglycerol hydrolase activity. The possible function of the liver in the serum triacylglycerol metabolism is discussed in connection with the proposed mechanism for the role of extrahepatic lipoprotein lipase in atherogenesis.