Osteopontin (OPN) is primarily a secreted phosphoglycoprotein found in a variety of tissues and body fluids. It has a wide range of reported functions, many of which are affected by the degree of post-translational modification (PTM) of the protein. These PTMs include phosphorylation, glycosylation, and cross-linking by transglutaminase. Here we describe the generation of unique monoclonal antibodies raised against recombinant OPN utilizing the OPN knockout mouse. The antibodies exhibit differential binding to OPN produced by different cell lines from the same species, as well to the multiple OPN forms in human urine. Most of the antibodies generated are able to recognize OPN produced by ras-transformed mouse fibroblasts, however only one antibody recognizes the more phosphorylated protein produced by the differentiating pre-osteoblast murine cell line MC3T3E1. Using a novel biopanning procedure combining T7 phage gene fragment display and protein G precipitation, we have epitope-mapped these antibodies. Several of the antibodies bind to regions of the OPN molecule that are phosphorylated, and one binds the region of OPN that is glycosylated. Using phosphorylated and non-phosphorylated peptides, we show that the binding of two antibodies to the C-terminal end of OPN is inhibited by phosphorylation of this region. In addition, these two antibodies are able to inhibit cell adhesion to recombinant and weakly modified OPN. The antibodies described herein may prove useful in determining the presence of modifications at specific sites and for identifying structural forms of OPN. Also, the sensitivity of these antibodies to PTMs suggests that caution must be taken when choosing anti-OPN monoclonal antibodies to detect this highly modified protein. J. Cell. Biochem. 102: 925–935, 2007. © 2007 Wiley-Liss, Inc.