We have found that the microsatellite marker AFM207zg5 (DXS995) maps to all previously described deletions which are associated with X-linked mixed deafness (DFN3) with or without choroideremia and mental retardation. Employing this marker and pHU16 (DXS26) we have identified two partially overlapping yeast artificial chromosome clones which were used to construct a complete 850 kb cosmid contig. Cosmids from this contig have been tested by Southern blot analysis on DNA from 16 unrelated males with X-linked deafness. Two novel microdeletions were detected in patients which exhibit the characteristic DFN3 phenotype. Both deletions are completely contained within one of the known DFN3-deletions, but one of them does not overlap with two previously described deletions in patients with contiguous gene syndromes consisting of DFN3, choroideremia, and mental retardation. Assuming that only a single gene is involved, this suggests that the DFN3 gene spans a chromosomal region of at least 400 kb.