The anchoring filament component laminin 5 (kalinin/nicein) is a candidate protein for mutations in some hereditary blistering skin disorders. In this study, laminin 5 expression was assessed in a family with generalized atrophic benign epidermolysis bullosa, a non-lethal valiant of the junctional form of epidermolysis bullosa. Immunofluorescence microscopy of the skin basement-membrane zone with a monoclonal antibody (GB3) revealed reduced anti-laminin 5 staining compared to normal controls. The labeling, when examined by immunoelectron microscopy, was present within the lower lamina lucida, immediately below the plane of blister formation. Numerous hemidesmosomes and well-formed anchoring filaments were seen on transmission electron microscopy. Polymerase chain reaction amplification of genomic DNA encoding the β3 subunit (LAMB3) of laminin 5, heteroduplex analysis of the polymerase chain reaction products, and nucleotide sequencing of the heteroduplexes revealed two putative mutations within the LAMB3 gene; these consisted of a premature termination codon in exon 3 and a mis-sense mutation in exon 7. Exons 3 and 7 encode part of domain VI of the laminin 5 β3 chain short arm. This globular domain of the protein has been postulated to have an important function in the interaction of laminin 5 with other structural components of the basement membrane zone, such as laminin 6 (K-laminin). Thus the mutations delineated in this family may have a critical pathogenetic significance in reducing adhesion between the epidermis and the dermis.