BACKGROUND. Osteopontin (OPN) is a secreted extracellular matrix (ECM) protein found in bone, as well as associated with epithelial cells. The main objective of these studies was to test in vitro the hypothesis that interaction with OPN stimulates proliferation of a quiescent subpopulation of prostate epithelial cells with high proliferative potential. METHODS. To simulate conditions that restrict proliferation and inhibit terminal differentiation of basal cells in vivo, control cultures grew on substrate coated with collagen (CO) or fibronectin (FN), in medium containing low levels of growth factors. RESULTS. Under growth-restricting conditions, most prostate epithelial cells with high proliferative potential, seeded in control secondary cultures, were quiescent within the time frame of the studies, as indicated by the small number of large colonies in these cultures. Growing prostate epithelial cells (PR) under the same growth- restricting conditions, but on substrate coated with OPN instead of CO or FN, stimulated proliferation of a subpopulation of single cells with high proliferative ability as indicated by: 1) dose-dependent increase in the percentage of single cells incorporating bromodeoxyuridine, i.e., proliferating PR; and 2) subsequent dose-dependent increase in the percentage of large colonies. The OPN effect was not merely due to preferential attachment to OPN, because PR attachment to OPN, CO, or FN was identical. PR attachment to OPN was inhibited in the presence of GRGDTP or an antibody against the integrin subunit α(v), but not in the presence of an RGES peptide or a nonspecific IgG. CONCLUSIONS. Integrin-mediated OPN/PR interaction stimulates proliferation of a quiescent subpopulation of prostate epithelial cells with high proliferative potential, possibly stem cells.