Osteopontin (OPN) is a highly modified protein that is found in many tissues and has been associated with a variety of physiological and pathological processes. Bone OPN is a potent inhibitor of hydroxyapatite crystal formation and stimulates bone resorption by osteoclasts; these activities, as well as others, are dependent upon phosphorylation of the protein. We have used mass spectrometry (MS) to perform a comprehensive analysis of the post-translational modification of OPN purified from rat bone. Matrix-assisted laser desorption time-of-flight (MALDI-TOF) MS showed masses of 37.6 and 36.8 kDa before and after enzymatic dephosphorylation, respectively, corresponding to a content of approximately 10.4 phosphate groups. Using proteolytic digestion and tandem MS, we localized 29 sites of phosphorylation: S10, S11, S46, S47, T50, S60, S62, S65, S146, T154, S160, S164, S167, S193, S196, S203, S220, S223, S232, S241, S245, S257, S262, S267, S278, S290, S295, S296, and S297. In addition, Y150 was shown to be sulfated and T107, T110, T116, and T121 are O-glycosylated. No glycan was detected at the potential N-glycosylation site. Other modifications, including deamidation, oxidation, and carbamylation, are also present. A 36-amino acid sequence from residues 67-102 could not be analyzed in detail, even after sialidase treatment, presumably because of the presence of a large number of acidic residues. In comparison to the previously characterized cow milk isoform, rat bone OPN is sulfated and has an additional site of glycosylation, many different sites of phosphorylation, and a lower overall phosphate content.