Platelet-type von Willebrand disease is a bleeding disorder resulting from gain-of-function mutations of glycoprotein (GP) Ibα that increase its affinity for yon Willebrand factor (vWf). The two known naturally occurring mutations, G233V and M239V, both enrich the valine content of an already valine-rich region within the Cys209-Cys248 disulfide loop. We tested the effect of converting other non-valine residues in this region to valine. Of 10 mutants expressed in CHO cells as components of GP Ib-IX complexes, four displayed a gain-of-function phenotype (G233V, D235V, K237V, and M239V) based on 125I-vWf binding and adhesion to immobilized vWf. The remainder displayed loss-of-function phenotypes. The gain-of-function mutants bound vWf spontaneously and had a heightened response to low concentrations of ristocetin or botrocetin, whereas the loss-of-function mutants bound vWf more poorly than wild-type GP Ibα. No distinct gain- or loss-of-function conforma-tions were identified with conformation-sensitive antibodies. Compared with cells expressing wild-type GP Ib3α, cells expressing the gain-of-function mutants rolled significantly more slowly over immobilized vWf under flow than wild-type cells and were able to adhere to vWf coated at lower densities. In aggregate, these data indicate that the region of GP Ibα bounded by Asn226 and Ala244 regulates the affinity for vWf.