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 beta 4 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 beta 4 subunit was absent in the skin of the PA-JEB patient, whereas the alpha 6 subunit appeared to be normally distributed along the basement membrane zone, as were the other hemidesmosomal components BP230, BP180 and HD1. Furthermore, the alpha 3 and beta 1 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 alpha 6 subunit. Like alpha 3, this subunit was colocalized with vinculin in focal contacts at the ends of actin stress fibers. Immunoprecipitation analysis revealed that alpha 6 was associated with beta 1 on PA-JEB keratinocytes, whereas normal human keratinocytes (NHKs) exclusively express alpha 6 beta 4 on their cell surface. The initial adhesion of PA-JEB and normal keratinocytes to laminin-1 and laminin-5, both ligands for alpha 6 beta 1 and alpha 6 beta 4, was similar. In migration assays, the PA-JEB keratinocytes were more motile on laminin-5 than normal keratinocytes. Our observations indicate that the integrin alpha 6 beta 4 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 beta 4 subunit, which results in the formation of too few and structurally abnormal hemidesmosomes.