The exact mechanism by which streptozotocin (STZ) destroys pancreatic β-cells has not been determined. Recently, nitric oxide was identified as a potent islet toxic compound. However, nitric oxide production by STZ has not been proved yet. In the present studies, we determined whether STZ spontaneously produces nitrite, measured by a spectrophotometric technique based on the Griess reaction. STZ, dissolved in the pH 4.0 citrate buffer, produced significantly more nitrite than that dissolved in pH 7.4. After incubation at 37°C for 120 min, nitrite concentrations were significantly decreased only in STZ dissolved in the pH 7.4 citrate buffer. Following 120 min incubation at 37°C, STZ dissolved at pH 7.4 failed to cause significant islet damage in rats previously deprived of food for 18 h. The present studies demonstrated that STZ may spontaneously produce nitrite in vitro. Nitric oxide production may, at least in part, be responsible for the STZ-induced damage of pancreatic β-cells of the rodents.