We have previously reported linkage of a large Finnish family with the generalized (Köbner) type of epidermolysis bullosa simplex to chromosome 12q in the region containing the type II keratin gene cluster (Ryynänen et al., Am J Human Genet 49:978-984, 1991). In this study, we examined the possibility that keratin 5, the type II keratin expressed in the basal keratinocytes, harbors the mutation in this family. Nucleotide sequencing revealed a T-to-C transition within exon 7 of the keratin 5 gene in the affected individuals of the family, while the unaffected individuals showed no evidence of C. The presence of the T-to-C transition in the affected individuals was confirmed by restriction enzyme digestion analysis with NciI endonuclease, as well as with PCR amplification of specific alleles (PASA) analysis. The PASA analysis also indicated that the mutated allele was not found among the 100 alleles tested within the general Finnish population indicating that the mutated allele is not a common polymorphism. Furthermore, the mutated allele was not present in nine individuals representing three different EBS families of Finnish origin. The T-to-C transition at the nucleotide level resulted in substitution of a leucine by a proline at the amino acid level, and the substitution affected a leucine residue which was invariant among eight different human keratins in a highly conserved segment at the carboxy-terminal region of the keratin 5 polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)