The adhesive glycoprotein vitronectin (VN) shows a high degree of conformational flexibility implicating that different molecular forms of the molecular may exist. Conformation-dependent monoclonal antibodies 13H1 or 16A7 that, per se, did not react with plasma VN bound to VN treated with heparin, chaotropes, detergents, pH below 6, or by heating at 56 degrees C. Dependent on the stimulus, recognition of VN by these antibodies varied and preceded heparin binding and self-association of VN resulting in the formation of noncovalently linked multimeric species of the protein. Both monoclonal antibodies also reacted with VN in serum or in platelet releasates as well as with VN in extracellular matrices of endothelial cells and inhibited cell adhesion on immobilized VN. Critical VN levels were needed for concentration-dependent multimerization indicating a nonlinear type of polymerization process. The nature of VN multimers was judged by nondenaturing gel electrophoresis, gel filtration, and sucrose gradient ultracentrifugation and revealed the formation of 3- to 16-mer multimeric species within an M(r) range of 200-1200 kDa representing a mean sedimentation coefficient of 9.6 S. In electron microscopy, multimeric VN occurred as globular specimens with an average diameter of 15-28 nm (monomeric plasma VN, 6-8 nm). In contrast to plasma VN, VN multimers were efficiently stabilized by covalent inter-molecular bonds following chemical or transglutaminase-induced cross-linking. A synthetic peptide comprising the central heparin binding region of VN (residues 348-361) not only bound to plasma VN but induced its multimerization also in plasma. During plasmin proteolysis of VN, fragments were generated that lacked the heparin binding region and that lost the ability to multimerize following urea or detergent treatment, implicating that the highly basic region is essential for multimer formation. These data suggest that non-plasma forms of VN, which are abundant in platelets and subendothelium, represent the prototype conformer of the reactive heparin binding form of VN. Our findings implicate that conformationally altered forms of VN enable the adhesive protein to multimerize in a characteristic fashion and thereby endow extracellular matrix sites with unique multivalent properties.