1. Hexokinase III was partially purified from the liver of the American bullfrong, Rana catesbeiana, using DEAE-cellulose column chromatography. 2. It was inhibited by glucose concentrations above 5 x 10(-5) M (pH 5.9), 10(-4) M (pH 6.7) or 10(-3) M (pH 7.5). 3. There was virtually no inhibition by excess glucose at pH 8.7. 4. The maximum velocity of the reaction increased with increasing pH. 5. Galactose could not be utilized as a substrate. 6. Classical Michaelis-Menten kinetics were obtained with respect to ATP, with no evidence of allostery. 7. The apparent Michaelis constant for ATP was 0.23 +/- 0.013 mM in the presence of 0.2 mM glucose at pH 7.5.