Nitric oxide (NO) has an important role in maintaining basal renal blood flow (RBF) and glomerular filtration rate (GFR) in the developing kidney. However, renal endothelial NO synthase (eNOS) has not been localized in the developing kidney. The purpose of this study was to examine the expression and localization of eNOS in the developing rat kidney using immunohistochemistry and western blotting. Kidneys from 14 (E14)-, 16 (E16)-, 18 (E18)- and 20-day-old (E20) fetuses, 1 (P1)-, 4 (P4)-, 7 (P7)-, 14 (P14)- and 21-day-old (P21) pups, and adult rats were extracted for immunohistochemistry, and western blot analysis. In the adult rat kidney, eNOS was expressed strongly in the endothelial cells of the arcuate artery and the vascular bundle in the medulla. Endothelial cells of the glomerulus and peritubular capillary network were weakly labeled for eNOS. There was no eNOS immunoreactivity in the uriniferous tubules, including the proximal tubules. In the developing rat kidney, eNOS appeared in the endothelial cells of the capillary network from E14. In the developing glomerular capillary, immunoreactivity for eNOS was observed in the S-shaped bodies (stage II glomeruli) and stage III glomeruli, whereas mature glomeruli (stage IV glomeruli) were faintly immunolabeled for eNOS. These eNOS-positive early-stage developing glomeruli were observed in the nephrogenic zone until seven days after birth. In the endothelial cells of the peritubular capillary network, eNOS was strongly expressed in the fetus and gradually decreased in intensity after birth. The endothelial cells of the arcuate artery were strongly immunoreactive for eNOS from E16 to the adult stages. In the renal medulla, eNOS was expressed in the endothelial cells of the capillary network surrounding the developing medullary collecting ducts of the fetal kidney. After birth, eNOS immunoreactivity gradually disappeared from the vasculature of the renal medulla and only remained in the vasa recta. In conclusion, the strong expression of eNOS in the early stages of the developing vasculature suggests that eNOS may contribute to angiogenesis and/or critically participate in the hemodynamics of the immature kidney.