In human liver, the oxidation of corticosteroids to 20-hydroxy-21-oic acids proceeds via the formation and oxidation of aldol (20-hydroxy-21-aldehyde) intermediates. Human liver aldehyde dehydrogenases E 1 and E 2, which we have previously purified to homogeneity, catalyzed the oxidation of steroid aldols by NAD + to 20-hydroxy-21-oic acids. The hydroxy acids formed after oxidation of the aldol isomer of cortisol (isocortisol) or of 11-deoxycorticosterone (isoDOC) by E 1 and E 2 respectively, were identified by the criteria of chromatographic mobility, derivatization, and reverse isotope dilution of 4- 14C labeled acid end products. Both enzymes showed broad substrate specificity and oxidized both 17-hydroxy and 17-deoxy steroids, though at widely varying rates. Kinetic analysis of the course of oxidation of isocortisol and isoDOC by NAD + gave intersecting initial velocity plots that conform with a sequential mechanism. The inhibition patterns for both enzymes with thionicotinamide adenine dinucleotide or chloral hydrate were consistent with random sequential behavior.