Prolonged exposure of pancreatic beta cells to the sulfonylureas glibencamide and tolbutamide induces subsequent desensitization to the actions of these drugs. The precise mechanisms underlying this desensitization remain unknown, prompting the present study, which investigated the impact of prolonged sulfonylurea exposure on glucose and energy metabolism using clonal pancreatic BRIN-BD11 beta cells. Following prolonged exposure to tolbutamide, BRIN-BD11 beta cells were incubated in the presence of [U-C-13]glucose, and isotopomer analysis revealed that there was a change in the ratio of flux through pyruvate carboxylase (EC 22.214.171.124) and pyruvate dehydrogenase (EC 126.96.36.199, EC 188.8.131.52, EC 184.108.40.206). Energy status in intact BRIN-BD11 cells was determined using P-31-NMR spectroscopy. Exposure to tolbutamide did not alter the nucleotide triphosphate levels. Collectively, data from the present study demonstrate that prolonged exposure of beta cells to tolbutamide results in changes in flux through key enzymes involved in glucose metabolism that, in turn, may impact on glucose-induced insulin secretion.