The pyruvate aldolases use pyruvate as the nucleophilic component in stereoselective aldol condensations, producing a 4-hydroxy-2-ketobutyrate framework. We have examined the 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolases from Pseudomonas putida, Escherichia coli, and Zymomonas mobilis for utility as synthetic reagents. Unlike other pyruvate aldolases examined to date, the KDPG aldolases accept short-chain, non-carbohydrate electrophilic aldehydes as substrates, providing a general methodology for the construction of the 4-hydroxy-2-ketobutyrate skeleton. The three aldolases differ markedly with respect to enzyme stability, pH optima, stability in organic cosolvent mixtures, substrate specificity, and diastereoselectivity during aldol condensation. All three enzymes show broad substrate specificity with regard to the electrophilic component. The primary requirements for substrate activity appear to be minimal steric hindrance and the presence of electron-withdrawing substituents at C2. The aldolases f...