Spectroscopic techniques (UV absorbance, circular dichroism, fluorescence emission and anisotropy, and light scattering) were used to investigate enzyme solubilization in Aerosol-OT (AOT) reversed micelles in which a bile salt, sodium taurocholate (NaTC), is used as a novel cosurfactant. NaTC significantly increases the water capacity and size of the reversed micelles through surfactant reorganization. The solubilization of several enzymes, including lysozyme, chymotrypsin, lipase, lipoxidase, carbonic anhydrase, and ribonuclease A, was demonstrated. These enzymes, ranging in mass from 10(4) to 10(5) Da, are incorporated in the micelles in stable, optically transparent solutions. Several other proteins were not successfully solubilized. The presence of NaTC in the reversed micelles significantly altered the conformations of the solubilized enzymes, apparently by promoting unfolding of the enzyme through interactions with the interior micellar interface. Lysozyme and lipase respond to solubilization in the AOT/NaTC micelles by altering their conformations to accommodate the micellar structure. The effect of NaTC is greatest far lysozyme, inducing a higher degree of order and helicity in the enzyme structure. Chymotrypsin, on the other hand, disrupts the micellar structure and reorganizes the surfactants to accommodate its own preferred conformation, Addition of NaTC to the reversed micelles causes a 3-fold increase in the enzymatic activity of solubilized chymotrypsin.