The high K_m glucose phosphorylation enzyme glucokinase is believed to be the α-cell glucose sensor, i.e., the site in glucose metabolism that determines the sensitivity and specificity of glucose-induced insulin secretion. We investigated the regulation of this enzyme by measuring glucokinase V_max and protein levels in isolated islets from hyperinsulinemic rats. Rats were infused for 48 h with 2 ml/h of 20% glucose, 50% glucose, or 0.45% NaCl (control rats). At the end of the infusion, 20% glucose-infused rats were normoglycemic and hyperinsulinemic (2.3-fold rise in basal plasma insulin level). Their islets had a 2.3-fold increase in insulin secretion at 8.3 mM glucose (51 ± 10% of capacity vs. 22 ± 5% in NaCl rats, P < 0.03), a 75% increase in glucokinase V_max and little if any increase in glucokinase protein level (111 ± 3% of control). The rats infused with 50% glucose had marked hyperglycemia and higher basal plasma insulin levels. Their islets were maximally stimulated by 8.3 mM glucose in combination with a 270% increase in glucokinase V_max and a 69 ± 11% increase in glucokinase protein level. Hexokinase V_max was also doubled. Thus, compensatory increases in β-cell glucose phosphorylation are a key mechanism for adaptive hyperinsulinemia. Our results show two types of regulation for the β-cell high K_m phosphorylation enzyme, glucokinase. The content of glucokinase protein is controlled by the plasma glucose level. Variable catalytic activity of this protein was also observed in this study.