In Escherichia coli, mannonic and altronic hydrolyases act, respectively, on mannonate, the intermediate aldonate of the glucuronate branch, and on altronate the intermediate aldonate of the galacturonate branch of the hexuronate pathway, yielding 2-keto-3-deoxy-gluconate. Our results demonstrate that the two hydrolyases are two distinct proteins. First, each hydrolyase shows a different induction pattern. In addition, separate constitutive mutants for either hydrolyase have been obtained. Second, single mutants negatively affected for one of the activities but not the other have been isolated in each case. Third, comparative heat inactivation of both activities at 59 degrees C shows mannonic hydrolyase to be clearly more thermosensitive than altronic hydrolyase. Furthermore the two enzymes also react differently to various effectors. Fourth, the two enzymes could be resolved on a DEAE cellulose column into two neighbouring but distinct peaks of activity, and a further purification yielded two pure hydrolyase fractions each being devoid of the activity of the other.