AbstractAttempts were made to demonstrate an adaptive increase in glucokinase in isolated perfused livers. Several physiological factors, such as insulin, hydrocortisone, high glucose and low fatty acid concentrations, which may have a regulatory effect on the adaptive processin vivowere tested. None of these were sufficient to bring about the adaptationin vitro. Glucokinase of livers from fasted rats and then perfused with either bicarbonate buffer, 100 % blood or with mixtures of these media were studied. The concentration of free fatty acids in the media was decreased by addition of insulin and elevated by anti‐insulin serum. These changes were not accompanied by change in glucokinase activity. A moderate decrease in glucokinase in livers from carbohydrate fed rats was observed when the livers were perfused for several hours with subphysiological glucose concentration in the medium. The enzyme was quite stable, however, when glucagon was added to the system. The adaptive increase in glucokinase activity could be demonstrated if the fasted rats were refed a carbohydrate diet for 3 hours before the livers were excised. However, addition of insulin to the perfusion medium was not necessary. When the protein synthesis inhibitor cyclohemixide was added to the perfusion system, the glucokinase from carbohydrate fed liver was quite stable whereas the enzyme in standard diet fed rat livers decreased significantly within 6 hours from the start of the perfusion. This indicates a different turnover rate of the enzyme under different physiological condition