Several metabolic compounds have been found to be competitive inhibitors of the anomerase activity of phosphoglucose isomerase (EC 5.3.1.9).Ki values for erythrose 4-phosphate, 6-phosphogluconate, and fructose 1,6-bisphosphate for the anomerase reaction are 0.32 muM, 21 muM, and 84 muM respectively at 0 degree and pH 8.2. A significant difference between the fructose 1,6-bisphosphate inhibition constants for both activities was found (Ki(isomerase) = 800 muM and Ki(anomerase) = 140 muM). Also the Km values for both activities were found to be significantly different (Km(isomerase) = 140 muM and Km(anomerase) = 3.6 muM). Attempts to independently alter the anomerase to isomerase activity ratio through protein modification yielded mixed results. While several modifying reagents destroyed the catalytic activities at identical rates, inactivation by iodoacetamide or pyridoxal 5' phosphate sensitized photo-oxidation displayed differential initial effects on the two activities with the anomerase activity being the less affected. These data support the theory that an imidazole residue is catalytically important for isomerization, but less so for anomerization.