Cultured mouse kidney cortical cells secrete osteopontin, a bone matrix protein that is also found in urine. Osteopontin is associated with cell proliferation/tumerogenesis and also inhibits kidney stone mineral crystal growth [1]. Using antibodies raised against osteopontin isolated from the culture medium, we localized osteopontin in normal rat kidney. Fluorescence, confocal and electron microscopy revealed osteopontin primarily in cells of the descending thin limb of the loop of Henle (DTL) and in papillary surface epithelium (PSE) in the area of the calyceal fornix. In situ hybridization with labeled RNA made from a cDNA that contains the entire coding sequence for mouse osteopontin revealed message at the same sites at which protein was demonstrated by immunocytochemistry. Immunogold labeling was localized to a population of dense vesicles distinct from lysosomes and endosomes. To examine the turnover of osteopontin, rats were injected with the protein synthesis inhibitor cyclohexamide, 14 mg/kg, six hours prior to kidney fixation. These kidneys no longer demonstrated osteopontin in DTL and the immunofluorescence in the papillary surface was attenuated. Thus, osteopontin is secreted at two sites in the kidney where urine is highly concentrated in stone mineral constituents. It has a relatively rapid turnover, suggesting that it could be subject to physiological regulation. Osteopontin may be important in the normal endogenous defense against kidney stone formation.