Osteopontin (OPN) is a secreted phosphoprotein expressed constitutively in the descending thin limb (DTL) and papillary surface epithelium (PSE) of the kidney. Although its function is not fully established, a role for OPN in the regulation of calcium-mediated or calcium-dependent processes has been proposed. The aim of this study was to examine the effects of 1,25-dihydroxyvitamin D<sub>3</sub> (vitD), a hormone involved in the regulation of calcium homeostasis, on renal OPN expression. Four groups of rats were studied: acute vehicle (single intraperitoneal [i.p.] injection of 0.1 ml 10% ethanol-90% propylene glycol, 12 h before being killed); acute vitD (single injection of vitD, 2 ng/g i.p., 12 h before being killed); chronic vehicle (daily subcutaneous [s.c.] injection of 0.1 ml 10% ethanol-90% propylene glycol for 7 days); and chronic vitD (daily s.c. injection of vitD, 0.5 ng/g, for 7 days). Kidneys were processed for light and electron microscope immunocytochemistry, in situ hybridization, and Western blot analysis. In vehicle-treated animals, OPN mRNA and protein were expressed primarily in the DTL and PSE. In the acute vitD group, OPN mRNA and immunoreactivity appeared in the thick ascending limb (TAL) of the inner stripe of the outer medulla, and increased slightly in the DTL and PSE. The proximal tubules exhibited strong OPN immunoreactivity, but no hybridization signal. In the chronic vitD group, there was a marked increase in OPN mRNA and immunoreactivity in the distal tubule, including the TAL, as well as in the DTL and PSE. A weak hybridization signal and immunostaining were also observed in some proximal tubules. Administration of vitD causes a marked increase in OPN mRNA and protein in the rat kidney, mainly in the distal nephron, but also in the DTL, PSE, and proximal tubules. These results indicate that vitD is involved in the regulation of OPN expression in the kidney.