Summary 1. Hexokinase and glucokinase activities were assayed in ultracentrifugally prepared fractions of livers and mammary glands of virgin, pregnant, lactating and 2-days-postlactating mice. 2. Liver contained both enzymes and both were located in the particle-free supernatant fraction. The levels of hepatic glucokinase activities observed in virgin, pregnant and postlactating mice were similar (about 600 mμmoles of TPNH formed per gram tissue), and about one-third of those observed in the lactating mice. 3. In the mammary glands of virgin mice glucose-ATP phosphotransferase activity was present mainly in the particle-free supernatant fraction. But in the glands of pregnant and lactating mice, glucose phosphorylation (solely by hexokinase) was also detected in the mitochondrial fraction. 4. The glands of virgin and pregnant mice contained both enzymes and, in contrast to liver, hexokinase activity exceeded that of glucokinase activity. Glucokinase was not detected in glands of lactating mice. Glucokinase activity in the virgin and pregnant glands probably reflects the presence of large amounts of adipose tissue in these glands. 5. The more than 2-fold increase in glucose phosphorylating activity observed in the mammary glands of lactating mice, as compared to that in the nonlactating mice, is attributed to the increase in activity of hexokinase associated with the mitochondrial fraction. This increase in hexokinase activity is probably related to the increased proportion of secretory elements of the lactating gland.