An analytical technique has been developed for the isoelectric focusing (IEF) of plasma von Willebrand factor (vWF) in agarose gels containing urea. Under these conditions, vWF freely enters the gel and focuses without artifact. The focused vWF is visualized by staining fixed gels with 125I-labeled affinity-purified heterologous antibody. Utilizing a pH gradient of 5.0-6.5, normal vWF in plasma or purified preparations focuses into at least three bands with apparent isoelectric points (pI) between pH 5.7 and 5.9. A reproducible difference in the IEF pattern of vWF has been established between normal plasmas and those of individuals with variant von Willebrand's disease (vWd) type IIA and type IIB. In type IIA, vWF has a distinctly lower pI than normal. This difference may be related to the presence of smaller vWF multimers in IIA plasma because forms of vWF of corresponding size contained in normal cryoprecipitate supernatant have a similar pI. Type IIB von Willebrand factor has a pI intermediate between normal and IIA. Neuraminidase treatment of plasma samples before IEF results in an increase in pI in normal, type IIA, and type IIB vWF. The data suggest that none of the 16 type IIA and 9 IIB plasmas studied here contain significantly decreased amounts of sialic acid.