OBJECTIVE In normal B-cells, glucokinase activity is regulated by glucose. We hypothesized that chronic exposure to low or high glucose levels would regulate glucokinase function and insulin secretion differently in islets of fa/fa compared with lean rats. SUBJECTS, DESIGN, and METHODS Islets isolated from lean and fa/fa rats (8-12 wk old) were cultured for 1-7 days in low (3.3 mM), moderate (12.5 mM) or supraphysiological (25.0 mM) glucose-supplemented medium. Sensitivity to glucose of hexokinase, glucokinase (by enzyme assay and kinetic analysis), and the insulin response (by radioimmunoassay) were assessed in each group of islets. RESULTS Islets of fa/fa rats cultured in 12.5 mM glucose for 1-7 days demonstrated a left-shift in both the EC50 of the insulin response and the Km of glucokinase to glucose. The glucokinase Vmax of fa/fa rat islets was lower under all conditions tested, thereby limiting the potential increase in insulin secretion. When cultured in 3.3 mM glucose for 1-7 days, fa/fa rat islets retained responsiveness to glucose longer and the estimated EC50 for glucose actually declined. However, the glucokinase Km for glucose increased three-fold in both phenotypes cultured in low glucose. Lean and fa/fa rat islets cultured in 25.0 mM glucose demonstrated a paradoxical hypersecretion of insulin to basal glucose concentrations and desensitization to stimulation by high concentrations of glucose. Islets from fa/fa rats were more easily desensitized, with significant effects in 25.0 mM glucose by 3 days compared with 7 days for the lean rat islets. Culture in high glucose erased the phenotype differences in glucokinase Km that were observed in 12.5 mM glucose cultured islets. CONCLUSIONS Differences in fa/fa rat islet glucokinase were observed only at moderate, near physiological glucose conditions. Glucokinase activity was similarly affected by low or high glucose in the two phenotypes, although differences in insulin secretion pattern were still detected, leading to the conclusion that factors other than glucokinase contribute to altered insulin secretion in the fa/fa rat. Further study of the glucose desensitization phenomenon in fa/fa rat islets might help unravel the factors that increase susceptibility to development of diabetes mellitus in some phenotypes.