Abstract: We describe a sensitive technique for measuring long-term islet cell proliferation rates in vivo in rats. Pancreatic islets were isolated and the incorporation of deuterium (2H) from heavy water (2H2O) into the deoxyribose moiety of DNA was measured by GC/MS. The results of heavy water labeling and BrdU staining were compared. The two methods were highly correlated (r = 0.9581, P < 0.001). Based on long-term heavy water labeling, ~50% of islet cells divided in rats between 8-15 weeks of age. Of interest, long-term BrdU administration suppressed proliferation of islet cells significantly, but not of bone marrow cells. Physiologic evidence further supported the validity of the method: older animals (24-weeks old) had 60% lower islet cell proliferation rates than younger rats (5-weeks old), and partial (50%) pancreatectomy increased proliferation by 20%. In addition, cholecystokinin-8 treatment significantly stimulated proliferation in pancreatectomized rats only. In summary, heavy water labeling is a quantitative approach for measuring islet cell proliferation and testing therapeutic agents.