Osteopontin is a secreted phosphoprotein that is expressed in the normal kidney and induced during various pathologic conditions associated with tubulointerstitial injury. However, the exact cellular location of osteopontin in the kidney has been a matter of controversy, and little is known about the role of osteopontin in the kidney. The purpose of this study was to establish the cellular and intracellular distribution of osteopontin in the rat kidney under normal conditions and after injection of a bacterial endotoxin lipopolysaccharide (LPS). Animals received injections of LPS or vehicle at different time intervals from 4 to 20 h before sacrifice. Kidneys were preserved by in vivo perfusion with paraformaldehyde-lysine-periodate (PLP) and processed for light and electron microscope immunocytochemistry using monoclonal antibodies to rat osteopontin. By light microscopy, immunostaining was observed in the descending thin limb and the papillary surface epithelium of both control and LPS-treated animals. After injection of LPS, osteopontin immunostaining was observed throughout the distal nephron and was also present in segments of the proximal tubule, where it was distributed in a punctate pattern. Staining was already present 4 h after injection of LPS and was maximal 6 h after injection. Electron microscopy revealed that osteopontin immunoreactivity in the descending thin limb and distal tubule cells was located in the Golgi apparatus and in small cytoplasmic vesicles, whereas in the proximal tubule labeling was observed in the vacuolar-lysosomal system. Western blot analysis demonstrated a band at approximately 70 kD and confirmed the increase in osteopontin expression after administration of LPS. These results demonstrate that osteopontin is constitutively expressed in cells of the descending thin limb and papillary surface epithelium and is induced throughout the distal tubule after administration of LPS.