We describe the isolation and purification of KDPGal aldolase from two sources. To facilitate this isolation, a facile five-step chemical synthesis of 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) (1) from commercially available D-galactono-1,4-lactone (3) is described. The key steps are the β- elimination of the protected aldonolactone 4 to furnish the desired 2,3- unsaturated ketone 5 and selective phosphorylation of the primary hydroxyl moiety of the diol 7. The purity of the KDPGal sample was > 95% as determined by enzymatic assay using crude KDPGal aldolase from Pseudomonas saccharophila. An initial substrate specificity and pH-activity profile of KDPGal aldolase are described.