The carbohydrate specificity of the two enzymes that catalyze the metabolic interconversions in the sorbitol pathway, aldose reductase and sorbitol dehydrogenase, has been examined through the use of fluoro- and deoxy-substrate analogs. Hydrogen bonding has been shown to be the primary mode of interaction by which these enzymes specifically recognize and bind their respective polyol substrates. Aldose reductase has broad substrate specificity, and all of the fluoro- and deoxysugars that were examined are substrates for this enzyme. Unexpectedly, both 3-fluoro- and 4-fluoro-D-glucose were found to be better substrates, with significantly lower K(m) and higher Kcat/K(m) values than those of D-glucose. A more discriminating pattern of substrate specificity is observed for sorbitol dehydrogenase. Neither the 2-fluoro nor the 2-deoxy analogs of D-glucitol were found to be substrates or inhibitors, suggesting that the 2-hydroxyl group of sorbitol is a hydrogen bond donor. The 4-fluoro and 4-deoxy analogs are poorer substrates than sorbitol, also implying a binding role for this hydroxyl group. In contrast, both 6-fluoro- and 6-deoxy-D-glucitol are very good substrates for sorbitol dehydrogenase, indicating that the primary hydroxyl group at this position is not involved in substrate recognition by this enzyme.