Although inducible nitric oxide synthase (iNOS) is known to play significant roles in the kidney, its renal localization has long been controversial. To resolve this issue, the authors identified iNOS-positive cell types in rat kidneys using double immunohistochemistry and confirmed iNOS positivity using enzyme histochemistry with NADPH-diaphorase (NADPH-d) and in situ RT-PCR. Adult male Sprague-Dawley rats were injected intraperitoneally with lipopolysaccharide (LPS) or saline as a control and sacrificed at various time intervals after injection. Quantitative real-time reverse transcriptase polymerase chain reaction showed that iNOS was not expressed in control kidneys but was induced in LPS-treated kidneys. iNOS immunostaining was strongest 6 to 18 hr after injection and decreased gradually to control levels by day 7. Double immunohistochemistry and NADPH-d revealed that iNOS expression was induced in the interstitial cells, glomerular parietal epithelial cells, the proximal part of the short-looped descending thin limb, the upper and middle papillary parts of the long-looped descending thin limb, some inner medullary collecting duct cells, and almost all calyceal and papillary epithelial cells. The present study determines the precise localization of iNOS in LPS-treated rat kidneys and provides an important morphological basis for examining the roles of iNOS in sepsis-induced acute kidney injury.