A permanent human hybrid endothelial cell line (EA.hy926) was shown to produce the von Willebrand factor, a protein of 250,000 relative mass (Mr) which was secreted into the medium as a 220,000 Mr protein. A cDNA library was constructed in lambda gt11 using mRNA from these hybrid cells. Several von Willebrand factor cDNA clones were isolated from this library using a synthetic oligodeoxyribonucleotide as a hybridization probe. These cDNA clones were used to analyze the von Willebrand factor gene in normal individuals and in cultured cells.