In post-ischemic acute kidney injury (AKI) or acute renal failure (ARF), dissipation of glomerular filtration pressure is associated with altered renal vascular tone and reactivity, as well as loss of vascular autoregulation. To test the hypothesis that renal nitric oxide (NO) generation reflects endothelial damage in the kidney after ischemia/reperfusion, we quantified urinary NO levels and identified the site of its generation in post-ischemic AKI. Subjects were 50 recipients of cadaveric renal allografts; 15 with sustained AKI and 35 with recovering renal function. Urine and blood samples were obtained after transplant, and intraoperative allograft biopsies were performed to examine nitric oxide synthases (NOS) in the kidney. In the sustained AKI group, urinary NOx (Nitrite and Nitrate) excretion, in µmol/g urine creatinine, was lower (12.3 ± 1.8 and 10.0 ± 1.4 on postoperative days 0 and 3) than in the recovery group (20.0 ± 3.6 and 35.1 ± 5.3* on postoperative days 0 and 3; * P < 0.005 vs. sustained AKI on day 0 and 3). eNOS expression diminished from peritubular capillaries of 6/7 subjects in the sustained ARF group, but from only 6/16 subjects in the recovery group. No differences were observed in iNOS staining pattern between the two groups. nNOS staining was rarely observed in macula densae of subjects, but was prominent in control tissues. These findings suggest that diminished NO generation by injured endothelium and loss of macula densa nNOS could impair the vasodilatory ability of the renal vasculature and contribute to the reduction in GFR in post-ischemic AKI.