Homogenates of rat pancreatic islets that had been heated for 5 min at 70 degrees C to inactive hexokinases, catalyzed the ATP-dependent phosphorylation of D-fructose. This reaction was dependent on the presence of K+ and was inhibited by D-tagatose although not by D-glucose or D-glucose 6-phosphate. The phosphorylation product was identified as fructose 1-phosphate through its conversion to a bisphosphate ester by Clostridium difficile fructose 1-phosphate kinase. These findings allowed the conclusion that fructokinase (ketohexokinase) was responsible for this process. Similar results were observed with tumoral insulin-producing cells (RINm5F line). Fructokinase may account for a large share of fructose phosphorylation in intact islets, particularly in the presence of D-glucose.