We have previously demonstrated that infusion of Intralipid to rats causes a pronounced increase of the lipoprotein lipase activity in the liver. In this paper we study where in the liver this lipoprotein lipase is located. When isolated livers from Intralipid-treated rats were perfused with heparin, substantial amounts of lipoprotein lipase were released into the perfusate. The identity of the lipase activity was demonstrated by specific inhibition with antisera to lipoprotein lipase, and to hepatic lipase, respectively, and by separation of the two lipase activities by chromatography on heparin-Sepharose. We have also studied the localization of both enzymes by an immunostaining procedure based on post-embedding incubation of ultrathin tissue sections with specific antibodies which were then visualized using protein A-colloidal gold complexes. There was no marked difference in localization for the two enzymes which were both seen at the luminal side of endothelial cells, at the interdigitations of the space of Disse and inside both hepatocytes and endothelial cells. Thus, lipoprotein lipase is present in the liver in positions similar to where the functional pool of hepatic lipase is located and analogous to where lipoprotein lipase is found in extrahepatic tissues. These results raise the possibility that the enzyme has a functional role in the liver.