Epidermolysis bullosa simplex (EBS) is a dominantly inherited genodermatosis characterized by intraepidermal blister formation. Recent reports have suggested that EBS mutations may relate to keratin abnormalities. In this study, we conducted RFLP analyses to test the hypothesis that EBS is linked to one of the keratin gene clusters on chromosome 12 or chromosome 17. Although these keratin gene loci are not defined by RFLPs, several mapped RFLPs in the same chromosomal regions could be tested for linkage. A large EBS family with 14 affected and 12 unaffected individuals in three generations was analyzed for RFLP inheritance. Within this family there was no evidence for linkage of the EBS mutation to markers on chromosome 17q. However, there was evidence for close linkage to D12S17 located on chromosome 12q, with a maximum LOD score of 5.55 at theta = 0. Mapping of this mutation to chromosome 12 defines an EBS locus distinct from both EBS1 (Ogna) and EBS2 (Koebner), which are on chromosomes 8 and 1, respectively. Further mapping will determine whether this EBS locus on chromosome 12 resides within the keratin gene cluster at 12q11-q13.