The structural similarities between the C-terminal domain of human pancreatic lipase (C-HPL) and C2 domains suggested a similar function, the interaction with lipids. The catalytic N-terminal domain (N-HPL) and C-HPL were produced as individual proteins, and their partitioning between the water phase and the triglyceride-water interface was assessed using trioctanoin emulsions (TC8). N-HPL did not bind efficiently to TC8 and was inactive. C-HPL did bind to TC8 and to a phospholipid monolayer with a critical surface pressure of penetration similar to that of HPL (15 mN m-1). These experiments, performed in the absence of colipase and bile salts, support an absolute requirement of C-HPL for interfacial binding of HPL. To refine our analysis, we determined the contribution to lipid interactions of a hydrophobic loop (β5′) in C-HPL by investigating a HPL mutant in which β5′ loop hydrophobicity was increased by introducing the homologous lipoprotein lipase (LPL) β5′ loop. This mutant (HPL-β5′LPL) penetrated into phospholipid monolayers at higher surface pressures than HPL, and its level of binding to TC8 was higher than that of HPL in the presence of serum albumin (BSA), an inhibitory protein that competes with HPL for interfacial adsorption. The β5′ loop of LPL is therefore tailored for an optimal interaction with the surface of triglyceride-rich lipoproteins (VLDL and chylomicrons) containing phospholipids and apoproteins. These observations support a major contribution of the β5′ loop in the interaction of LPL and HPL with their respective substrates.