The structural transitions of the tetrameric rabbit muscle pyruvate kinase induced by guanidine hydrochloride and urea are characterized by elastic and quasi-elastic light-scattering, sedimentation velocity, and intrinsic viscosity experiments as well as by protein fluorescence, circular dichroism, and enzymic activity measurements. The transition curves are shown to be reversible. We find a new pathway of unfolding which is different from that described in the literature: The first intermediate with increasing concentration of denaturant is a less compact and inactive tetramer which can be renatured if substrates are added. Dissociation of the tetramer results in an expanded dimer with a partial loss of the secondary structure. The final state is a completely disordered monomer. These intermediates are consistent with a domain structure of pyruvate kinase, as it was suggested by Stammers & Muirhead [Stammers, D. K., & Muirhead, H. (1975) J. Mol. Biol. 95, 213--225] on the basis of their X-ray data. Using Schellman's solvent denaturation model [Schellman, J. A. (1978) Biopolymers 17, 1305--1322], we calculate the free energies of stabilization of the folding--unfolding equilibrium.