Junctional epidermolysis bullosa (JEB) comprises a group of inherited autosomal recessive blistering disorders characterized by dermo-epidermal separation through the lamina lucida of the basement membrane. We identified a patient with JEB associated with pyloric atresia (PA), in whom the integrin β4 subunit was completely absent. At the ultrastructural level, the hemidesmosomes were reduced in number, appeared rudimentary and lacked a subbasal dense plate and frequently an inner attachment plaque. However, keratin filaments were still anchored to the cytoplasmic plaque of the hemidesmosome. Immunofluorescence analysis showed that the β4 subunit was absent in the skin of the PA-JEB patient, whereas the α6 subunit appeared to be normally distributed along the basement membrane zone, as were the other hemidesmosomal components BP230, BP180 and HD1. Furthermore, the α3 and β1 subunits were not only detected at the lateral membranes of basal cells in PA-JEB skin, as in normal skin, but also along the basement membrane zone. The few hemidesmosome-like structures found in cultured keratinocytes from the PA-JEB patient contained the hemidesmosomal components BP230, BP180 and HD1, but not the integrin α6 subunit. Like α3, this subunit was colocalized with vinculin in focal contacts at the ends of actin stress fibers. Immunoprecipitation analysis revealed that α6 was associated with β1 on PA-JEB keratinocytes, whereas normal human keratinocytes (NHKs) exclusively express α6β4 on their cell surface. The initial adhesion of PA-JEB and normal keratinocytes to laminin-1 and laminin-5, both ligands for α6β1 and α6β4, was similar. In migration assays, the PA-JEB keratinocytes were more motile on laminin-5 than normal keratinocytes. Our observations indicate that the integrin α6β4 plays a crucial role in the proper assembly of hemidesmosomes and in the stabilization of the dermal-epidermal junction. The fragility of the skin and the blistering in this patient appear to have been due to the deficiency of the integrin β4 subunit, which results in the formation of too few and structurally abnormal hemidesmosomes.