2-Keto-3-deoxy-6-phosphogluconate aldolases (KDPG aldolases, EC 4.1.2.14) from four sources, Esherichia coli, Pseudomonas putida, Pseudomonas saccharophila, and Zymononas mobilis, were purified from 17- to 48- fold by differential dye-ligand chromatography (DD-L chromatography). This technique, using Procion Navy H-ER (150 grain) as a negative column and Procion Yellow MX-GR as a positive column supported on Sepharose CL-4B, represents a general methodology for the rapid, facile purification of KDPG aldolases from disparate microbial sources. © 1994.