Chromatography of bovine kidney aldose reductase using Matrex Orange A affinity gel results in the separation of the unactivated and activated enzyme forms. The former washes through the column, while the latter is eluted with an NADPH step-gradient. The separated enzyme forms display V max and K m glycolaldehyde values, and relative sensitivities to inhibition by the aldose reductase inhibitor AL-1576 (spiro[2,7-difluorofluorene-9, 4′-imidazolidine]-2′,5′-dione), that are similar to those reported previously for the individual forms. However, because V max is 17-fold lower for the unactivated enzyme, the purification of aldose reductase via NADP(H) elution from a dye-ligand affinity matrix can result in the selective purification of only the activated enzyme form. These results have direct implications for the study of potential aldose reductase inhibitors, and may explain why linear double-reciprocal plots are commonly observed for enzyme prepared in this manner, while nonlinear plots are seen in other cases.