It has been suggested that branchio-oculo-facial (BOF) syndrome, deafness with ear pits and other similar conditions [MIM #125100, 120502], and branchio-oto-renal (BOR) [MIM #113650] or Melnick-Frazer syndrome represent different phenotypes of the BOR syndrome. The latter is inherited in an autosomal dominant (AD) manner and has an extremely variable clinical expression. Recently, the BOR gene was mapped to chromosome 8q13.3 and its sequence was identified as the human homolog of the Drosophila eyes absent(EYA1) gene. We studied an extended family with AD inheritance of branchial arch anomalies (BAA), neurosensory hearing loss and ear pits; their phenotype resembled that of a family described by Fourman et al. (BMJ 1955;2:1354) and differed from patients with BOR, in that none of the affected members had renal abnormalities or lacrimal duct stenosis. Fifteen affected members were studied; ear pits were present in all of them (100%), whereas hearing loss and other BAA were present in 33% and 10%, respectively. Blood was collected from 31 members of this large family. DNA was extracted from peripheral blood lymphocytes by standard methods, and PCR-amplified using primers from microsatellite sequences flanking the BOR locus on chromosome 8q13.3 (D8S1807, D8S530, and D8S543). Linkage analysis was performed using the LINKAGE programs under two models of AD inheritance with different penetrance: 100% and 80%. In both cases, the produced LOD scores were significantly less than -2; exclusion of the 8q13.3 locus was also confirmed by multipoint LOD score analysis. We conclude that, at least in one large family with AD inheritance of BAA, hearing loss and ear pits, the BOR locus was excluded. BAA are present in genetically heterogeneous syndromes; the identification of the responsible genes will lead to better understanding of craniofacial development and the genetics of hearing.