The platelet GPIIb-GPIIIa heterodimer (integrin _IIb3) binds fibrinogen with high affinity in response to activation by agonists, leading to platelet aggregation and formation of a hemostatic plug. The ³²⁶GRV motif in GPIIb is highly conserved in the -subunit of other integrins, suggesting that it might play an important functional role. Moreover, Arg³²⁷His substitution in GPIIb has been associated with defective platelet surface expression of GPIIb-IIIa and thrombasthenic phenotype. This work aimed at elucidating whether the absence of Arg³²⁷ or its substitution by His was responsible for the impaired surface expression of GPIIb-IIIa complexes. Transfection of cDNA encoding [Ala³²⁷]GPIIb, [Gln³²⁷]GPIIb, or [Phe³²⁷]GPIIb into Chinese hamster ovary cells inherently expressing GPIIIa permitted surface exposure of GPIIb-IIIa complexes, whereas [Glu³²⁷]GPIIb did not. These observations indicate that it is not the loss of [Arg³²⁷]GPIIb but the presence of His³²⁷ or a negatively charged residue like Glu at position 327 of GPIIb that prevents the surface exposure of GPIIb-IIIa heterodimers. In contrast, changing Gln³⁴⁴, the homologue to Arg³²⁷ in the -subunit of the vitronectin receptor, to His did not prevent the surface expression of _v-GPIIIa complexes. Thus the conformational constraint imposed by His³²⁷ seems to be rather specific for the heterodimerization and/or processing of GPIIb-IIIa complexes.