The folding of two monofunctional fragments of aspartokinase-homoserine dehydrogenase I has been studied. One of these fragments corresponds to the kinase activity and the N-terminal part of the polypeptide chain; the other one corresponds to the dehydrogenase activity and to the C-terminal part of the chain. Both fragments are able to refold into an enzymatically active conformation after complete disruption of their native structure. The kinase fragment folds up into an active monomeric species. The dehydrogenase fragment first folds up into an inactive monomeric species and then associates into an active dimeric species. These two fragments thus correspond to regions capable of autonomous folding. The folding of each of these fragments is compared to that of the corresponding region in the intact aspartokinase--homoserine dehydrogenase I reported previously [Garel, J.R., & Dautry-Varsat, A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3379-3383]. It is concluded that the N-and C-terminal regions of the intact polypeptide chain behave as independent folding units. A model of the sequence of steps involved in the folding process of aspartokinase-homoserine dehydrogenase I is presented; its relevance to the evolution of this protein is also discussed.