The chromosomal region around marker D8S277 is thought to contribute to susceptibility to hearing impairment in patients with the 1555A>G mutation in mtDNA. We have previously described a family with this mutation, in which some of the members had profound hearing loss, some had a hearing impairment for high-frequency tones and some had completely normal hearing. The phenotypes were thus compatible with a recessive inheritance pattern. We fine-mapped the region around marker D8S277 by sequencing single nucleotide polymorphisms (SNPs) along the 11 Mb region on 8p23, and also sequenced eight defensin genes in the vicinity of D8S277 and the genes GJB2, GJB3, MTO1 and TIMM8A. SNP haplotypes were constructed using the SimWalk2 program. The three persons with a profound hearing loss had identical genotypes in the 11 Mb region on 8p23, but this genotype was also present in a person with normal hearing. The persons with a hearing impairment for high-frequency tones did not share any common haplotype, but one of them shared a genotype with a healthy person. Thus, haplotype comparison excluded a contribution of the region concerned to the expression of hearing impairment in this family, nor could the susceptibility be assigned to the GJB2, GJB3, MTO1 or TIMM8A genes. Extended pedigrees with 1555A>G, such as the present one, provide a good opportunity to identify a modifying nuclear factor. The chromosomal region around 8p23 could be excluded here as the locus for susceptibility to hearing impairment.