In the range of guanidine hydrochloride concentrations from 0.2 to 1.2 M, aspartokinase-homoserine dehydrogenase I loses its enzymatic properties, both kinase and dehydrogenase activities and their allosteric inhibition by L-threonine. Ligands which stabilize the tetrameric native structure protect the enzyme against inactivation. Under some conditions, all the functional properties do not disappear at the same rate: an intermediate species possessing only the kinase activity can be detected. Several arguments suggest that this partly active intermediate has a monomeric structure. These results show that deactivation of aspartokinase-homoserine dehydrogenase I is a stepwise process, compatible with the reverse of the previously described reactivation [Garel, J.-R., & Dautry-Varsat, A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3379-3383]. The same measurements performed with a monofunctional fragment carrying the dehydrogenase activity show that the loss of dehydrogenase activity is the same whether or not the polypeptide chain is intact or lacks the kinase region; this finding suggests that the protein is composed of independent regions. The influence of protein aggregation in studying unfolding-refolding of oligomeric enzymes is also discussed.