To evaluate the biosynthetic basis for decreased glycoprotein (GP) Ib-IX expression resulting from GP IX mutations described in three siblings with Bernard-Soulier syndrome, we introduced each mutation into the cDNA for GP IX by site-directed mutagenesis (GP IX Asp21 --> Gly and GP IX Asn45 --> Ser) and examined the associations of the mutants with the two other subunits of the GP Ib-IX complex in transfected cells. Unlike wild-type GP IX, neither of the mutants was able to increase GP Ib expression on the cell surface, either when transfected into Chinese hamster ovary (CHO) alpha beta cells or when cotransfected with GP Ib alpha and GP Ib beta into wild-type CHO cells. We also evaluated whether cotransfecting wild-type or mutant GP IX with GP Ib beta would result in the appearance of GP IX on the surface of the transfected cells; the wild-type protein was detected on the surface of the cells, whereas neither mutant reached the cell surface in appreciable quantities. Immunofluorescence microscopy of permeabilized cells revealed that the failure to express mutant GP IX on the cell surface did not result from failure to synthesize the polypeptide. Both mutants were detected in intracellular compartments, albeit at lower levels than the wild-type polypeptide (the fluorescence of cells expressing the GP IX Asp21 --> Gly was consistently the lowest). Direct evidence that the mutants associate poorly with Gp Ib beta was obtained of 35S-labeled cells transiently expressing GP Ib beta and wild-type or mutant GP IX. The amount of GP IX coprecipitated with GP Ib beta was greatly diminished in cells expressing either mutant. These findings suggest an important role for the conserved leucine-rich motif of GP IX in the association of this polypeptide with GP Ib beta and provide further evidence for the importance of GP IX in the stability of the GP-Ib-IX complex.