Vitronectin (VN), secreted into the bloodstream by liver hepatocytes, is known to anchor epithelial cells to basement membranes through interactions with cell surface integrin receptors. We report here that VN is also synthesized by urothelial cells of urothelium in vivo and in vitro. In situ hybridization, dideoxy sequencing, immunohistochemistry, and ELISA of urothelial cell mRNA, cDNA, tissue, and protein extracts demonstrated that the VN gene is active in vivo and in vitro. The expression of VN by urothelium is hypothesized to constitute one of several pathways that anchor basal cells to an underlying substratum and explains why urothelial cells adhere to glass and propagate under serum-free conditions. Therefore, two sources of VN in the human urinary bladder are recognized: 1) localized synthesis by urothelial cells and 2) extravasation of liver VN through fenestrated capillaries. When human plasma was fractionated by denaturing heparin affinity chromatography, VN was isolated in a biologically active form that supported rapid spreading of urothelial cells in vitro under serum-free conditions. This activity was inhibited by the matricellular protein SPARC via direct binding of VN to SPARC through a Ca(+2)-dependent mechanism. A novel form of VN, isolated from the same heparin affinity chromatography column and designated as the VN(c) chromatomer, also supported cell spreading but failed to interact with SPARC. Therefore, the steady-state balance among urothelial cells, their extracellular milieu, and matricellular proteins constitutes a principal mechanism by which urothelia are anchored to an underlying substrata in the face of constant bladder cycling.