To exploit the enzymatic method for the synthesis of beta-hydroxy-alpha-amino acids, the genes coding for the Escherichia coli L-threonine aldolase (LTA; EC 2.1.2.1) and Xanthomonus oryzae D-threonine aldolase (DTA) were cloned and overexpressed in E. coli through primer-directed polymerase chain reactions. The purified recombinant enzymes were studied with respect to kinetics, specificity, stability, additive requirement, temperature profile, and pH dependency. DTA requires magnesium ion as a cofactor, while LTA needs no metal ions. These enzymes work well in the presence of DMSO with concentration up to 40%, and DMSO-induced rate acceleration of LTA-catalyzed reaction was observed. Both enzymes use pyridoxal phosphate coenzyme to activate glycine to react with a wide range of aldehydes. LTA gave erythro-beta-hydroxy-alpha-L-amino acids with aliphatic aldehydes and the three isomer with aromatic aldehydes as kinetically controlled products. On the other hand, DTA formed threo-beta-hydroxy-alpha-D-amino acids as kinetically controlled products with aliphatic and aromatic aldehydes but the diastereoselectivity was lower than that of LTA. Under optimal conditions, several P-hydroxy-a-amino acid derivatives (3-hydroxyleucines, gamma-benzyloxythreonines, gamma-benzyloxymethylthreonines, and polyoxamic acids) have been stereoselectively synthesized on preparative scales using these enzymes. Also, the tandem use of DTA and phosphatases has made possible the synthesis and separation of D-allo-threonine phosphate and D-threonine.