Mutations in DFNA5 cause a non-syndromic autosomal dominant type of hearing loss. Although not much is known regarding the physiological function of DFNA5, it is not only related to hearing loss. A clear link with cancer exists. For example, methylation of the 5' flanking region of DFNA5 was detected in breast, colorectal and gastric cancer. So far, this 5' flanking region has not been studied in detail. Here, we describe the identification of the cochlear transcription initiation site (TIS), the identification of the core promoter region between -120 and +70 relative to the TIS and the identification of an enhancer (between -121 and -356 bp) and a silencer element (between -356 and -670 bp). Results were similar in HEK293 cells and in the organ of Corti cell line OC-k3. Transfection with a reversely-oriented construct resulted in high transcriptional activity. We subsequently confirmed this antisense activity and identified a novel antisense transcript partly overlapping Dfna5.