—There is indirect evidence for a gender difference in nitric oxide (NO) synthesis from vascular endothelium. The aim of the present study was to determine NO production more directly in healthy women and men by the measurement of ¹⁵N nitrate excreted in urine after the intravenous administration of l-[¹⁵N]₂-guanidino arginine. Twenty-four healthy volunteers (13 men aged 22 to 40 years and 11 women aged 23 to 42 years) participated in this study. No subjects were receiving any medication. Women were studied between the 7th and 14th days of their menstrual cycles. Arterial blood pressure was measured oscillometrically, and 1.13 μmol l-[¹⁵N]₂ arginine was administered intravenously after an overnight fast. Urine was collected for the next 36 hours in separate 12-hour periods. Urinary ¹⁵N/¹⁴N nitrate ratio was assessed by dry combustion in an isotope ratio mass spectrometer. Mean 36-hour urinary ¹⁵N nitrate excretion was greater in women than in men (2111±139 versus 1682±87 ηmol; P<0.05). Furthermore, total urinary ¹⁵N nitrate excretion was associated inversely with the mean arterial blood pressure in the whole group of subjects (coefficient of correlation, 0.47; P=0.022). The present data show that whole-body production of NO is greater in healthy premenopausal women than in men under ambulatory conditions. The cellular origin of NO measured in this study is unknown, but differences in endothelial production could underlie differences in vascular function between men and women.