Human pancreatic lipase (HPL) loses more than 80% of its activity when incubated with tetrahydrolipstatin in a buffer containing bile salts. During the inactivation process, large changes are observed in intrinsic tryptophan fluorescence and in the near-ultraviolet circular dichroism. The rate of chemical inactivation is highly comparable to that determined from the time dependence of the spectral changes. It is concluded that HPL undergoes a conformational transition upon inhibitor binding, resulting in a change in the microenvironment of tryptophan residues. Bile salts are needed in this system for effective inactivation of the enzyme by tetrahydrolipstatin, and a large increase in the inactivation rate takes place at about the critical micellar concentration (CMC) of bile salts. The inhibited enzyme can be reactivated by reducing the bile salt concentration to below the CMC, and the changes in tryptophan fluorescence induced by acylation with tetrahydrolipstatin are thereby reversed. This suggests that bile salts above their CMC stabilize the acyl-enzyme complex.