Two hexokinase activities of different substrate specificity (fraction I and II) have been isolated by means of column chromatography on DEAE-cellulose from the antennal gland of the crayfish Orconectes limosus (RAFINESQUE). Chromatographically, the two fractions are uniform. Out of 10 sugars, fraction I converts mannose and fructose only; the phosphorylation of fructose amounts to 20% only of the conversion of mannose. Fraction II phosphorylates six out of the 10 sugars tested, and in its substrate specificity it corresponds to an unspecific hexokinase. The products of phosphorylation are with both enzymes for all substrates sugar-6-phosphates. The Km values of fraction I for mannose and fructose are 3,9·10-5 and 2,9·10-3Μ, those of fraction II for glucose, mannose and fructose 7,1·10-5Μ, 8,0·10-5Μ and 6,7·10-3Μ. The same enzymes have been found in the heart and in the hindgut, with proportions in the three organs, however, different. Experiments with the crude extract had the following results as regards the properties of the enzymes: the activity is not particle-bound. Optimal activity is obtained at a pH-value of more than 7.7. High concentrations of salt as well as pH-values below 6 or above 8 rapidly destroy the activity in storage.