Exogenous bilirubin (BR) substitutes for the protective effects of heme oxygenase (HO) in several organ systems. Our objective was to investigate the effects of exogenous BR in an in vivo model of ischemia-reperfusion injury (IRI) in the rat kidney. Four groups of male Sprague-Dawley rats were anesthetized using isoflurane in oxygen and treated with 1) 5 mg/kg intravenous (iv) BR, 1 h before ischemia and 6-h reperfusion; 2) vehicle 1 h before ischemia and 6-h reperfusion; 3) 20 mg/kg iv BR, 1 h before and during ischemia; and 4) vehicle 1 h before and during ischemia. Bilateral renal clamping (30 min) was followed by 6-h reperfusion. Infusion of 5 mg/kg iv BR achieved target levels in the serum at 6 h postischemia (31 ± 9 μmol/l). Infusion of 20 mg/kg BR reached 50 ± 22 μmol/l at the end of ischemia, and a significant improvement was seen in serum creatinine at 6 h (1.07 ± 28 vs. 1.38 ± 0.18 mg/dl, P = 0.043). Glomerular filtration rate, estimated renal plasma flow, fractional excretion of electrolytes, and renal vascular resistance were not significantly improved in BR-treated groups. Histological grading demonstrated a trend toward preservation of cortical proximal tubules in rats receiving 20 mg/kg iv BR compared with control; however, neither BR dose provided protection against injury to the renal medulla. At the doses administered, iv BR did not provide complete protection against IRI in vivo. Combined supplementation of both BR and carbon monoxide may be required to preserve renal blood flow and adequately substitute for the protective effects of HO in vivo.