Chronic exposure of rat pancreatic islets and INS-1 insulinoma cells to glucosamine (GlcN) produced a reduction of glucose-induced (22.2 mM) insulin release that was associated to a reduction of ATP levels and ATP/ADP ratio, compared to control groups. To further evaluate mitochondrial function and ATP metabolism, we then studied uncoupling protein UCP-2, F₁- F₀ATP-synthase and mitochondrial membrane potential, a marker of F₁-F₀ATP-synthase activity. UCP-2 protein levels were unchanged after chronic exposure to glucosamine. both on pancreatic islets and INS-1 cells, Due to the high number of cells required to measure mitochondrial F₁- F₀ATP-synthase protein levels and mitochondrial membrane potential, we used INS-1 insulinoma cells, and we found that chronic culture with GlcN increased F₁-F₀ATP-synthase protein levels but decreased glucose-stimulated changes of mitochondria membrane potential. Moreover, F₁-F₀ATP-synthase was highly glycosylated, as demonstrated by experiments with N-glycosydase F and glycoprotein staining. Tunicamycin (an inhibitor of protein N-glycosylation), when added with glucosamine in the culture media, was able to partially prevent all these negative effects on insulin secretion, adenine nucleotide content, mitochondrial membrane potential and protein glycosylation. Thus, we suggest that GlcN-induced pancreatic -cell toxicity might be mediated by reduced cell energy production. An excessive protein N-glycosylation of mitochondrial F₁-F₀ATP-synthase might lead to cell damage and secretory alterations in pancreatic -cell.