Heat exposure early in ovine pregnancy results in placental insufficiency and intrauterine growth restriction (PI-IUGR). We hypothesized that heat exposure in this model disrupts placental structure and reduces placental endothelial nitric oxide synthase (eNOS) protein expression. We measured eNOS protein content and performed immunohistochemistry for eNOS in placentas from thermoneutral (TN) and hyperthermic (HT) animals killed at midgestation (90 days). Placental histomorphometry was compared between groups. Compared with the TN controls, the HT group showed reduced delivery weights (457 +/- 49 vs. 631 +/- 21 g; P < 0.05) and a trend for reduced placentome weights (288 +/- 61 vs. 554 +/- 122 g; P = 0.09). Cotyledon eNOS protein content was reduced by 50% in the HT group (P < 0.03). eNOS localized similarly to the vascular endothelium and binucleated cells (BNCs) within the trophoblast of both experimental groups. HT cotyledons showed a reduction in the ratio of fetal to maternal stromal tissue (1.36 +/- 0.36 vs. 3.59 +/- 1.2; P< or = 0.03). We conclude that eNOS protein expression is reduced in this model of PI-IUGR and that eNOS localizes to both vascular endothelium and the BNC. We speculate that disruption of normal vascular development and BNC eNOS production and function leads to abnormal placental vascular tone and blood flow in this model of PI-IUGR.