To understand the pathophysiology of junctional epidermolysis bullosa (JEB), a disease characterized by blister formation at the lamina lucida level of the cutaneous basement membrane, we analyzed the expression and assembly of laminin-5 in patients' keratinocytes. Previously we showed that the laminin-5 α3β3γ2 heterotrimer assembles intracellularly via a β3γ2 heterodimer intermediate. We were interested in how the heterogeneous group of defects in laminin-5 found among different JEB patients may effect, or block, the assembly and secretion of a complete laminin-5 heterotrimer, and whether the severity of the clinical phenotype might correlate with the degree to which laminin-5 chain assembly is interferred with. We analyzed a variety of patients, so for example, in one patient with a less severe form of the disease, a truncated β3 chain participated in heterotrimer formation that resulted in otherwise normal assembly and secretion. In two other patients with life threatening forms of JEB, defects in the β3 chain result in an absence of assembly intermediates, β3γ2 heterodimer and β3 monomer, due to only limited expression of the β3 chain. A small amount of normal β3 chain was detectable that was rapidly assembled into heterotrimers that were secreted. The most severe phenotype may have been of a patient who expired from JEB within 9 weeks from birth. In this patient with lethal disease, there was a complete absence of the α3 chain. Although the assembly intermediates, β3γ2 heterodimers and β3 and γ2 monomers were present, no heterotrimers could be assembled, and none could be secreted. Interestingly, fibronectin expression was apparently increased generally in JEB patient cells as compared to normals. Fibronectin may serve as an alternative attachment molecule for cells defective in laminin-5, and its overexpression may be an attempt to partially compensate for the genetic defect in laminin-5. We conclude that the extent of complete laminin-5 heterotrimer assembly and secretion in JEB keratinocytes correlates well with the clinical prognosis.