The versatility of integrin functions is mediated by engagement of a number of proteins that assemble with integrins. Among them, paxillin is one of the important molecules interacting with a variety of signaling molecules and cytoskeletal building blocks. We report here that paxillin is not a single molecule with a unique physiological property. We identified two human paxillin isoforms, beta and gamma. These isoforms have distinct amino acid insertions; each consists of a distinct exon, at the same site of previously reported paxillin (paxillin alpha). Several proteins were co-precipitated with paxillin, and we found that beta bound to focal adhesion kinase but weakly to vinculin, and gamma bound to vinculin but only weakly to focal adhesion kinase, although both bound equally to talin. No additional proteins were found to bind to beta and gamma over those binding to alpha. Unlike the alpha isoform, beta and gamma mRNAs were not detected in normal tissues, but several cancer cells expressed both alpha and beta proteins simultaneously. All three isoform proteins were expressed in promonocytic cells with ratios comparable with each other, and the expression patterns were altered during differentiation of floating promonocytic cells into adherent macrophage-like cells. Therefore, each isoform of paxillin exhibits distinct expression and different biochemical as well as physiological properties and thereby appears to act as a distinct module involved in different functions of integrins.