G lucose regulates the cellular content of glucokinase in the pancreatic beta cell by altering the level of the enzyme. We investigated the existence of a second regulatory pathway, an alteration in the catalytic activity, by comparing Vmax and protein levels of glucokinase in rat islets cultured under high glucose conditions (16.7 mM) for 6, 14, and 24 h. The Vmax was increased by glucose at all time points. In contrast, glucokinase protein levels on Western blots were unchanged from the control value at 6 h but increased 40% at the later time points (P < 0.0002). Further evidence for a dual regulatory system was obtained with a reversal protocol. After a 6-h incubation at high glucose, an additional 3-h incubation at 5.5 mM glucose restored glucokinase Vmax to normal, but failed to change the Vmax after a 24-h incubation at high glucose. Finally, 10 microM cycloheximide partially prevented the increase in glucokinase Vmax induced by 24 h of high glucose, but had no effect at 6 h, suggesting the early increase in enzymatic activity did not require protein synthesis. In summary, glucose regulates both the catalytic activity and cellular content of glucokinase in the beta cell. Glucose-induced increases in glucokinase activity are an important element of the beta cell adaptive response to hyperglycemia.