Osteopontin (OPN) is a secreted protein that has been implicated in diverse physiological and pathological processes. OPN can bind to integrins, via GRGDS or SVVYGLR amino acid sequences, and to other cell surface receptors, and many of OPN's functions are likely mediated via cell adhesion and subsequent signaling. Here we developed and characterized a series of five monoclonal antibodies, raised to distinct internal peptide sequences of human OPN, and have used these sequence‐specific reagents, along with the previously described anti‐OPN monoclonal antibody mAb53, to map functional epitopes of OPN that are important to cell adhesion and migration. All antibodies were reactive with native as well as recombinant human OPN. One antibody (2K1) raised against the peptide VDTYDGRGDSVVYGLRS could inhibit RGD‐dependent cell binding to OPN, with an efficacy comparable to that of mAb53. Furthermore, 2K1 could inhibit α9 integrin‐dependent cell binding to OPN. The epitope recognized by 2K1 was not destroyed by thrombin digestion, whereas mAb53 has been shown to be unable to react with OPN following thrombin cleavage. The two distinct epitopes defined by 2K1 and mAb53 antibodies are closely related to the SVVYGLR cell‐binding domain and the GLRSKS containing thrombin cleavage site, respectively, and are involved in cell binding and cell migration. J. Cell. Biochem. 84: 420–432, 2002. © 2001 Wiley‐Liss, Inc.