Osteopontin (OPN) is a phosphorylated glycoprotein that occurs in high levels in bone, but is also present at sites of pathological calcification. Based on autotitration, OPN is regarded as a potent inhibitor of hydroxyapatite crystal formation, an activity that requires phosphorylation of the protein. Vitamin K is also a stone inhibitor, which has an effect on vitamin K-dependent gamma-glutamyl carboxylase activity. To explore the mechanism, using different doses of vitamin-K, we developed an enzyme-linked immunosorbent assay (ELISA) using a combination of mouse monoclonal antibodies against OPN in the rat kidney. We also investigated the expression of OPN mRNA in the kidney in an experimental model of a renal stone, using mass spectrometry to identify 27 serines and two threonines that are phosphorylated in the stone isoform of OPN.