The molecular defect of a new Bernard-Soulier patient, originating from Morocco and presenting thrombocytopenia with large platelets and an absence of ristocetin-induced platelet agglutination, has been identified and reproduced in transfected heterologous cells. Gene sequencing revealed insertion of a guanine in the domain coding for the transmembrane region of the glycoprotein (GP) Ib beta subunit. This mutation causes a translational frame shift, which creates putative novel transmembrane and cytoplasmic 37 and 125 amino acids domains, respectively. A 34 kDa immunoreactive GPIb beta band, instead of the normal 26 kDa subunit, was detected by Western blotting in lysates from the patient's platelets and from transfected cells and in immunoprecipitates of metabolically labeled cells. The abnormal subunit did not associate with GPIb alpha and was mainly intracellular, although a significant fraction could reach the cell surface. Cells expressing the mutant GPIb-IX complex adhered to a von Willebrand factor matrix but were unable to change shape, unlike cells expressing the wild-type receptor. These results strongly suggest a novel role of the GPIb beta subunit and its transmembrane-intracellular region in GPIb-VWF-dependent signaling, in addition to a role in correct assembly and cell surface targeting of the GPIb-V-IX complex.