The knowledge about gene mutations causing permanent hearing impairment (HI) is rapidly increasing, offering clinicians the possibility of analysing different gene mutations in relation to various phenotypes. This study examines a possible relationship between U-shaped audiograms and mutations in the GJB2-gene, coding for Connexin 26 (Cx 26). Thirty-eight subjects at a median age of 42 years, range 18-60 years with symmetric U-shaped audiograms classified as sensorineural were included in the genetic investigation. The gender distribution was 13 males and 25 females. No subjects had any indication of syndromic HI, and any possible exogenous factor that might cause HI was excluded. Three subjects had self-reported prelingual HI and 34 subjects had self-reported postlingual HI. Thirty-five subjects had one or more family members with HI. In 19 subjects the entire Cx 26 gene was examined, whereas 19 subjects were investigated for the 35delG mutation only. One female with mild HI and postlingual onset of the HI was heterogeneous for the L9OP-mutation in the Cx 26 gene. In all other subjects no mutations in the Cx 26 gene could be identified. Mutations of the Cx 26 gene are very rare among subjects exhibiting a U-shaped phenotype of the audiogram. However the majority of the investigated subjects (35/38) had a family history of HI and it seems therefore reasonable to ascribe U-shaped hearing deficit to genetic factors which has to be searched for in alternative gene mutations.