We reported previously that plasma levels, urinary excretion, and metabolic production of cyclic guanosine 3′,5′‐monophosphate (cGMP) are increased in gravid rats, and postulated that endogenous nitric oxide (NO), a potent vasodilator and immune modulator, may mediate this change. Four lines of evidence are now presented demonstrating increased biosynthesis of NO during pregnancy in rats: 1) Urinary excretion and plasma levels of the stable NO metabolite, nitrate, are elevated in pregnant rats; urinary excretion of nitrate is increased in pseudopregnant rats. 2) The urinary excretion of cGMP also increases during pregnancy and pseudopregnancy, paralleling the rise in urinary nitrate excretion. 3) Chronic treatment with the NO synthase inhibitor, N^G‐nitroarginine methyl ester (NAME), inhibits the increase in urinary nitrate excretion. 4) Nitric oxide hemoglobin is detected by electron paramagnetic resonance spectroscopy in the blood of pregnant, but not in nonpregnant, rats. The results show endogenous NO production is increased in gravid rats. This finding raises the possibility that NO may contribute to maternal vasodilation and uterine immune suppression of normal pregnancy.—Conrad, K. P., Joffe, G. M., Kruszyna, H., Kruszyna, R., Rochelle, L. G., Smith, R. P., Chavez, J. E., Mosher, M. D. Identification of increased nitric oxide biosynthesis during pregnancy in rats. FASEB J. 7: 566‐571; 1993.