Herlitz junctional epidermolysis bullosa (H-JEB) is characterized by hampered expression of the adhesion ligand laminin-5. Thus far, analysis of the processes underlying the epithelial-mesenchymal dysadhesion marking this disease has been limited by the reduced growth and adhesive capabilities of the epithelial cells derived from H-JEB patients. To overcome these difficulties, we used SV40 virus to immortalize H-JEB keratinocytes with a homozygous nonsense mutation in the gene that encodes the gamma2 chain of laminin-5. Cell lines (LSV) derived from infected keratinocytes maintain a stable karyotype, grow independent of 3T3 feeder layers and are not tumorigenic. Further analysis of clone LSV5 showed an increased secretion of laminin-6 and fibronectin compared to normal keratinocytes. Similar to parental H-JEB keratinocytes, these cells regenerate stratified epidermis in vitro and, in in vivo models, they synthesize a basement membrane lacking laminin-5. LSV cells show hypermotility and reduced adhesive properties resulting from an incomplete association with the underlying culture substrate. These results demonstrate that LSV5 cells retain the pathologic phenotype of H-JEB keratinocytes and can serve as a model system to study the adhesion processes mediated by laminin-5.