A unique and heretofore undescribed glycoprotein with unusual properties has been purified and characterized from the culture medium of endothelial cells. This protein is synthesized constitutively by bovine, porcine, and human endothelial cells, by vascular smooth muscle cells, and by fibroblasts from dermis and ligament. It is also a biosynthetic product of some murine malignant and/or transformed cell lines but was not uniformly observed in cells derived from human neoplasms. The glycoprotein exhibited an apparent molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 39,000 before reduction, and of approximately 43,000 (43K protein) in the presence of dithiothreitol. Amino acid analysis revealed high levels of potentially acidic residues (Asx + Glx = 303 residues/1000) and of cysteine (35 residues/1000). Limited proteolysis indicated that both disulfide bonds and mannosylated sites were distributed throughout the protein chain. Neither phosphate nor sulfate was incorporated into the 43K protein during biosynthetic labeling of endothelial cells. In addition, the 43K protein did not bind to heparin, thrombin, gelatin, or fibronectin and displayed no affinity for [3H]diisopropyl fluorophosphate. In contrast, the 43K protein demonstrated a high affinity binding to bovine serum albumin which was dissociable only by sodium dodecyl sulfate. A complete lack of identity with several prominent serum and platelet proteins and with other mesenchymal cell products was shown by one- and two-dimensional peptide mapping, affinity chromatography, and immunological studies. Immunofluorescence staining of endothelial cells showed a granular distribution for the 43K protein that was typical of a secreted protein. The function of this apparently novel glycoprotein is presently not known. Its synthesis by normal mesenchymal cells and by malignant or transformed cells of both ectodermal and endodermal origin suggests a general role in cell function that is independent of transformation.