A rare mutation in Brn-3c (Brn3.1, POU4F3) underlies adult onset hearing loss (DFNA15) and targeted deletion of this gene in mice leads to complete deafness due to loss of sensory hair cells from the cochlea. Therefore the aim of our study was to identify and characterise common functional variation in the Brn-3c gene, which could potentially be a genetic risk for more common forms of adult onset hearing loss. We identified seven sequence variants at the Brn-3c locus. One of these, a novel, common variant at position -3432 was extremely complex consisting of a variable guanine repeat that also exhibited single nucleotide substitutions within the poly-guanine repeat: -3432 poly-G polymorphism. In-vitro studies show that this polymorphism modifies binding affinity for the SP1 transcription factor. Furthermore, reporter constructs of the Brn-3c 5'-flanking region containing different -3432 poly-G alleles show altered transcriptional activity when endogenous SP1 levels are reduced using a dominant negative approach. Results also indicate that this effect is influenced by the length of a novel polymorphic (GT)(n) repeat at position -566 in the Brn-3c 5'-flanking region. In summary, our data show there are common sequence variants in the Brn-3c 5'-flanking region that affect transcriptional regulation in vitro; these variants are candidates for large-scale population based association analysis as they could potentially affect the genetic risk for more common types of adult onset hearing loss.