The D-glucose anomeric preference of hexokinases partially purified from animals (rat, mouse, and chicken) and baker's yeast (Saccharomyces cerevisiae) were investigated by the assay system with glucose-6-phosphate dehydrogenase as a coupling enzyme. With low Km hexokinases in animal tissues and cells, the ratios of Vmax for the beta-anomer to Vmax for the alpha-anomer (V beta/V alpha) were within a range from 1.3 to 1.5. In yeast, the V beta/V alpha value was 1.1 for hexokinase A, 0.8 for hexokinase B, and 1.4 for glucokinase. The possible explanation for D-glucose anomeric preference of hexokinase is discussed.