Endothelium-derived nitric oxide (NO) is synthesized within the developing kidney and may play a crucial role in the regulation of renal hemodynamics. The purpose of this study was to establish the expression and intrarenal localization of the NO-synthesizing enzyme endothelial NO synthase (eNOS) during kidney development. Rat kidneys from 14 (E14)-, 16 (E16)-, 18 (E18)-, and 20-day-old (E20) fetuses and 1 (P1)-, 3 (P3)-, 7 (P7)-, 14 (P14)-, and 21-day-old (P21) pups were processed for immunocytochemical and immunoblot analysis. In fetal kidneys, expression of eNOS was first observed in the endothelial cells of the undifferentiated intrarenal capillary network at E14. At E16, strong eNOS immunoreactivity was observed in the endothelial cells of renal vesicles, S-shaped bodies (stage II glomeruli), and stage III glomeruli at the corticomedullary junction. At E18-20, early-stage developing glomeruli located in the subcapsular region showed less strong eNOS immunoreactivity than those of E16. The eNOS-positive immature glomeruli were observed in the nephrogenic zone until 7 days after birth. In fetal kidneys, eNOS was also expressed in the medulla in the endothelial cells of the capillaries surrounding medullary collecting ducts. After birth, eNOS immunostaining gradually increased in the developing vascular bundles and peritubular capillaries in the medulla and was highest at P21. Surprisingly, eNOS was also expressed in proximal tubules, in the endocytic vacuolar apparatus, only at P1. The strong expression of eNOS in the early stages of developing glomeruli and vasculature suggests that eNOS may play a role in regulating renal hemodynamics of the immature kidney.