In the presence of electron acceptors rabbit muscle aldolase catalyzes the oxidation of dihydroxyacetone phosphate and in the course of this activity the enzyme is progressively inactivated [Christen, P., Cogoli-Greuter, M., Healy, M. J., and Lubini, D. (1976) Eur. J. Biochem. 63,223- 2311. We propose that the agent responsible for inactivation is the highly electrophilic a-ketoaldehyde produced in the reaction. This ketoaldehyde is herein shown to react rapidly with arginine sidechains, forming stable adducts, and with an active center residue in aldolase, leading to inactivation of the enzyme. Substrate-dependent oxidative inactivation of aldolase is brought about by reaction in situ of hydroxypyruvaldehyde phosphate with the essential residue located in the active site. The role of nascent hydroxypyruvaldehyde phosphate in aldolase inactivation is also borne out by studies with the substrate analogue D-5-ketofructose 1,6-bisphosphate. Cleavage of this hexose bisphosphate by aldolase yields hydroxypyruvaldehyde phosphate in the absence of any oxidant and, as in the case of oxidative production of the ketoaldehyde, the catalytic cycles lead to inactivation of the enzyme. A similar role is suggested for nascent ketoaldehydes in the substrate-dependent oxidative inactivation of class I1 aldolase of yeast and transaldolase. Possible physiological significance of a-ketoaldehyde production is discussed. Carbanionic enzyme-substrate intermediates are susceptible to oxidation : several enzymes forming such intermediates were shown to catalyze the oxidation of their substrates in the presence of suitable electron acceptors [l]. The carbanion intermediate of aldolase-dihydroxyacetone phosphate complex is also oxidized by a number of redox indicators [1,2], dihydroxyacetone phosphate being converted to hydroxypyruvaldehyde phosphate. Concomitant to this unusual ‘dehydrogenase’ activity aldolase is inactivated [3]. The action of the inactivating agent was found to be practically restricted to the enzyme molecule that has produced it, suggesting that it is a transiently reactive entity. It was therefore concluded that an oxidatively activated intermediate, tentatively identified as a free radical, is responsible for the covalent modification and inactivation of aldolase [3]. This paper presents evidence for a role of hydroxypyruvaldehyde phosphate in the inactivation of aldolase. This ketoaldehyde reacts with arginine residues