Integrin beta 4 is expressed primarily within the epithelial basement membranes, and it contributes to the stable association of epidermis with the underlying basement membrane. Previous observations have suggested that the expression of this integrin, which is alternatively spliced in various cell types, is deficient in a variant of junctional epidermolysis bullosa associated with pyloric atresia (JEB-PA). To facilitate identification of mutations in the human beta 4 integrin gene, ITGB4, we have now determined its intron-exon organization. The entire gene was shown to consist of 41 exons spanning 36 kb of the genomic DNA on chromosome 17q11-qter. The mutation detection, using PCR-amplification of each exon followed by heteroduplex analysis, revealed a homozygous splicing mutation in a patient with JEB-PA. RT-PCR revealed the presence of two splice variants generated through utilization of cryptic splice sites, and both mRNA transcripts resulted in a frame-shift and premature termination codon of translation. The presence of the mutation resulted in dramatic reduction of the corresponding mRNA transcript level. Because beta 4 integrin is expressed not only in the skin but also in the epithelial lining of the stomach, the absent expression of this integrin in the proband may explain the blistering tendency and development of pyloric atresia.