The structure of human serum albumin (HSA) in the pressure-induced denatured state was investigated by fluorescence spectroscopy. HSA undergoes a conformational change in the pressure range from 0.1 MPa to 400 MPa, at 25 degrees C. Several ligands bind to specific sites in HSA, and the fluorescence spectra of these ligands were used to study the conformational state of this protein. The warfarin-binding site (site I) and the dansylsarcosine-binding site (site II), are located in subdomains II and III, respectively. The fluorescence spectra of these probes reflected the structural changes in each of these subdomains. Dansylsarcosine completely dissociated from its binding site in domain III above 300 MPa, but substantial affinity of warfarin remained in this pressure range. Similar results were obtained for the urea-induced denaturation of HSA; although dansylsarcosine completely dissociated at urea concentration above 6 M, warfarin remained bound to site I in domain II at these concentrations. These results suggest that the structure of domain III is unfolded both in the initial stages of both pressure- and urea-induced denaturation of HSA. HSA possesses a single tryptophan residue (Trp-214) in domain II, and fluorescence from this residue reflects structural changes in this domain. In the urea-induced denatured state of HSA, a red-shift in the wavelength of maximum fluorescence occurred over urea concentrations ranging from 4 M to 6 M. This shift indicated that a structural change in domain II occurred simultaneously with the unfolding of domain III in this concentration range. On other hand, the shift in the wavelength of maximum fluorescence of Trp-214 was comparatively small in the pressure range from 0.1 MPa to 400 MPa indicating that the environment of Trp-214 was not affected. These results indicate that preferential unfolding of domain III occurs in the pressure-induced denatured state of HSA.