Prolonged intermittent exposure to subanesthetic concentrations of nitrous oxide (N2O) can impair spermatogenesis in the LEW/f mai rat. USP purity standards state that N2O used for medical purposes may contain other oxides of nitrogen such as NO and NO2 as impurities at concentrations up to one part per million (ppm) each. The question thus arises as to whether prolonged exposure to N2O may be associated with adverse health effects from oxides of nitrogen other than N2O, particularly NO2. NO2 has demonstrated biological toxicity at concentrations as low as 0.3 ppm. In this study, rats were exposed to filtered air with and without 1 ppm NO2 for 7 hr/day, 5 days/week, for 21 days. No gross or light microscopy abnormality was found in the testes of NO2-exposed rats. Serum vitamin B12 levels were within normal limits. The findings indicate that NO2 exposure under the test conditions does not in itself impair spermatogenesis or alter B12 levels.