Interfacial catalysis of hepatic triacylglycerol lipase (H-TGL) and lipoprotein lipase (LpL) isolated from human post-heparin plasma was investigated with mixed monolayers of trioleoylglycerol (TO) and egg phosphatidylcholine. Rates of enzyme catalysis were dependent on surface pressure, substrate concentration, apoC-II (the activator protein for LpL), and cholesteryl oleate (CO). LpL showed a surface pressure optimum between 22 and 24 mN m-1, whereas H-TGL activity decreased at pressures greater than 20 mN m-1. LpL activity was enhanced greater than 10-fold by apoC-II; 1 M NaCl inhibited enzyme activity. ApoC-II, apoC-III, apoA-I, apoA-II, and 1 M NaCl had no effect on H-TGL activity. The substrate (TO) dependency was different for the two lipases. For LpL, there was a marked enhancement of enzyme activity between 2 and 4 mol % TO, whereas for H-TGL, enzyme activity increased linearly between 1 and 10 mol % TO. LpL activity toward monolayers containing 2 mol % TO was enhanced 2.6-fold by the addition of 5 mol % CO; cholesteryl ester had no effect on H-TGL activity. These findings suggest that the two lipolytic enzymes have different interfacial properties, which may have relevance to the rates of hydrolysis of triacylglycerols at a lipoprotein interface.