Although osteopontin (Opn) is a strong inhibitor of calcium oxalate crystallization in vitro, its role in stone formation in vivo is unknown. We investigated the renal expression of Opn in normal, ethylene glycol (EG), and EG + ammonium chloride-treated rats. Male Sprague-Dawley rats were divided into three groups. Group 1 (N = 5) was the control. Animals in Group 2 (N = 6) received 4 weeks of treatment with 0.75% EG, and Group 3 animals (N = 6) were given 1 week of treatment with 0.75% EG and 1.0% NH4Cl. The kidneys were then examined for crystal deposition and Opn localization. In normal rats, staining for Opn was evident in the proximal tubules (PT; S3 > S2 > S1), distal tubules (DT), and the thick ascending limbs of Henle (TAL) and a few collecting ducts (CD). All rats in Group 3 had significant crystal deposition throughout their kidneys. In Group 2 rats, Opn staining increased in all segments of the PT, DT, and TAL. Staining in these tubular segments was even greater in Group 3 rats, including the CD and the papillary surface epithelium. In addition, Opn was present within all crystal deposits. Renal Opn expression in experimental urolithiasis becomes stronger and more diffuse as the severity of the lithiasis-inducing treatment increases. These results are consistent with the hypothesis that renal epithelial cells produce larger amounts of osteopontin to combat the development of kidney stones.