Endotoxin-related acute kidney injury (AKI) has been shown to profoundly induce nitric oxide (NO) which activates sympathetic and renin-angiotensin system resulting in renal vasoconstriction. While vascular muscle cells are known to upregulate inducible nitric oxide synthase (iNOS), less is known about the endothelium as a source of NO during endotoxemia. Studies were therefore undertaken both in vitro in mouse microvascular endothelial cells and in vivo in transgenic mice with overexpression of endothelial GTP cyclohydrolase, the rate limiting enzyme for BH₄, a cofactor for NOS. LPS significantly induced endothelial cell iNOS expression and NO concentration in the culture media with no change in eNOS expression. GTPCH transgenic (Tg) mice demonstrated a significant increase in baseline urine NO/creatinine ratio and a more significant increase in renal iNOS expression and serum NO levels with LPS treatment as compared to the WT mice. GFR and RBF decreased significantly in Tg mice with LPS 1.0mg/kg while no changes were observed in WT with the same dose of LPS. Serum IL-6 levels were significantly higher in Tg as compared to WT mice during endotoxemia. The antioxidant, tempol, improved the GFR in the Tg mice. Thus, endothelium can be an important source of iNOS and serum NO concentration during endotoxemia, thereby increasing the sensitivity to AKI. Reactive oxygen species appear to be involved in this acute renal injury in Tg mice during endotoxemia.