A detailed investigation concerned with localizing hexokinase in the Novikoff ascites tumor is presented. At least 50% of the total hexokinase activity was shown by differential and density gradient centrifugation techniques to be associated with tumor mitochondria. None of this activity was latent. Fractionation of isolated tumor mitochondria with digitonin revealed an outer membrane location for this enzyme. Treatment of tumor mitochondria with glucose 6-phosphate released about 80 to 85% of the hexokinase activity without disrupting the intermembrane compartment. This suggests that at least this proportion of the activity is bound to the outer surface of the outer membrane. Successive treatments did not remove the remaining hexokinase activity. At 30 degrees C, an incubation time of about 10 min with glucose 6-phosphate was required to achieve maximal release. No solubilization occurred at 0-4 degrees C. The isozymes derived from Novikoff mitochondria were identified by anion exchange chromatography as types I and III. Glucokinase activity was not detectable. Evidence is also presented which indicates that the hexokinase obtained from Novikoff mitochondria binds to the outer membrane of rat liver mitochondria. In contrast, the low endogenous hexokinase activity present in isolated liver mitochondria was found not to fractionate with outer membrane markers, but rather with contaminating microsomal membrane markers. Results described here provide the first direct evidence for the submitochondrial location of hexokinase in a tumor. They reveal an outer membrane location and an involvement of two hexokinase isozymes. Because these findings are characteristic of the hepatoma and not observed in control liver preparations, it is suggested that they may be very relevant to the general property of rapidly growing tumors to catabolize large amounts of glucose.