Epidermolysis bullosa (EB) is a group of heritable mechano-bullous skin diseases classified into three major categories on the basis of the level of tissue separation within the dermal-epidermal basement membrane zone. The most severe, dystrophic (scarring) forms of EB demonstrate blister formation below the cutaneous basement membrane at the level of the anchoring fibrils. Ultrastructural observations of altered anchoring fibrils and genetic linkage to the gene encoding type VII collagen (COL7A1), the major component of anchoring fibrils, have implicated COL7A1 as the candidate gene in the dystrophic forms of EB. We have recently cloned the entire cDNA and gene for human COL7A1, which has been mapped to 3p21. In this study, we describe mutations in four COL7A1 alleles in three patients with severe, mutilating recessive dystrophic EB (Hallopeau-Siemens type, HS-RDEB). Each of these mutations resulted in a premature termination codon (PTC) in the amino-terminal portion of COL7A1. One of the patients was a compound heterozygote for two different mutations. The heterozygous carriers showed an approximately 50% reduction in anchoring fibrils, yet were clinically unaffected. Premature termination codons in both alleles of COL7A1 may thus be a major underlying cause of the severe, recessive dystrophic forms of EB.