Background: Pregnancy is associated with an upregulation of nitric oxide in the uteroplacental unit reported to be due to increased activity of consitutive nitric oxide synthase (cNOS) activity. Aim: To measure nitric oxide production directly in the utero-placental unit and evaluate the potential of inducible NOS (iNOS) as the enzyme source.Methods: Timed-pregnant rats were sacrified on day 21 of a 22 day gestation. NO release was determined in uterus and placenta explants in tissue culture media (RPMI 1640), within minutes of sacrifice using a electrochemical technique. NO flux was detected as a change of the current from baseline with a NO sensitive microelectrode. iNOS immunohistochemistry and gene expression were measured in uterus and placentas. Similar studies were performed in non-pregnant females. Results: Using RT-PCR, no iNOS gene expression was detected in the non-pregnant uterus. With pregnancy, iNOS was expressed using both RT-PCR and immunohistochemistry (placental-sinusoids; uterus-smooth muscle). NO release bordered on the limits of detection of the microelectrode(<50nM) in the non-pregnant uterus (cNOS only). With pregnancy, NO release was increased to 119±17 and 633±41nM in the uterus and placenta, respectively. Conclusions: These results demonstrate that iNOS gene expression in pregnancy is associated with a marked increase in NO release. We postulate that iNOS, and not cNOS, is the primary source of NO in the pregnant utero-placental unit.