The distinguishing feature of lipases is their increased activity at an oil-water interface, termed interfacial activation. X-ray crystallography of lipases suggested a mechanism for interfacial activation by revealing conformational changes in several surface loops that cover the active site. In one conformation, these loops prevented substrate from entering the active site, and, in the other conformation, movement of the loops opened the active site. We tested the role of the major surface loop, the lid domain, in human pancreatic lipase (hPL) function by creating deletions in this region and expressing the mutant proteins in baculovirus-infected insect cells. the mutants were tested for activity against tributyrin and triolein, colipase interaction, interfacial activation, and binding to tributyrin. The purified mutants had decreased activity against both tributyrin and triolein compared to wild-type hPL and did not show a preference for either substrate. Although colipase was required for maximum activity in the presence of bile salts, the mutants had significant activity against tributyrin, but not triolein, in the absence of colipase. Both mutants were active against monomers of tributyrin demonstrating that they did not require an interface for activity. Finally, both mutants had decreased binding to tributyrin particles. These results suggest that the lid domain in hPL mediates interfacial activation and influences interfacial binding.