Hepatic triglyceride lipase was obtained from post-heparin plasma of rats in an electrophoretically homogeneous form. The enzyme had an isoelectric point at pH 4.9 and molecular weight of 65,000. The relation between the "lipase" and "esterase" activities of the enzyme was studied using emulsified triolein and water-soluble methyl butyrate (80 mM) as substrates. The same enzyme protein catalyzed the hydrolyses of both emulsified triolein and water-soluble methyl butyrate. The relation of activity to the methyl butyrate concentration differed from those for pancreatic lipase and liver esterase. During purification, the ratio of methyl butyrate to triolein-hydrolyzing activity of the enzyme increased. On digestion of the enzyme with trypsin, the "lipase" activity was retained. However, the trypsin-treated enzyme was adsorbed to a heparin-Sepharose column and eluted with 0.75 M NaCl, like the untreated enzyme. These results suggest that rat hepatic triglyceride lipase contains a so-called "hydrophobic recognition site" that is destroyed by trypsin treatment and is distinct from the heparin-binding and catalytic sites.