Twenty-seven stable subclones of seven independent cellular hybrids producing murine monoclonal antibodies to human von Willebrand factor (vWF) have been established. The specificity of the hybridoma antibodies for vWF has been substantiated by a variety of methods including binding to highly purified vWF, absence of binding to plasma or cryoprecipitate from severe von Willebrand's disease, binding to different size multimers in normal plasma, and binding to low molecular weight multimers in type IIA von Willebrand's disease plasma. Monoclonality of the hybridoma derived antibodies has been sought by repeated cloning at limiting dilutions. All seven specificities of hybridoma antibodies cross-react to a variable degree with porcine vWF and all but one with bovine vWF, indicating that they bind to structural loci that are relatively though not identically conserved between species. Hybridoma antibodies bind native as well as denatured vWF, suggesting that the epitopes may be determined to a considerable extent by primary structure rather than entirely by tertiary or quaternary conformation. The hybridoma antibodies define precise epitopes in the vWF molecule, extend the horizon of direct analysis of vWF to the intramolecular level, and may be useful in assigning functional loci to parts of the molecule.