The cellular location of hepatic lipase was investigated in transgenic rabbits that expressed human hepatic lipase in the liver. The binding of monoclonal antibodies to human hepatic lipase, as detected by either fluorescence-tagged or gold-conjugated secondary antibodies, showed that hepatic lipase was concentrated at the surfaces of hepatic sinusoids. This distribution was the same as observed in the human liver. At the ultrastructural level, immunogold labeling of the space of Disse showed hepatic lipase on both lumenal and sublumenal surfaces of rabbit liver sinusoidal endothelial cells. An equivalent amount of hepatic lipase also was found on the external surfaces of hepatocyte microvilli in the space of Disse, as well as in the interhepatocyte spaces. The distribution suggests that a majority of the hepatic lipase produced by the liver is associated with hepatocyte surfaces, consistent with the functions of this enzyme in lipoprotein metabolism.