The connexins are a family of at least 20 homologous proteins in humans that form aqueous channels connecting the interiors of coupled cells and mediating electrical and chemical communication. Mutations in the gene for human connexin 31 (hCx31) are associated with disorders of the skin and auditory system. Alterations in functional properties of Cx31 junctions are likely to play a role in these diseases; nonetheless, little is known about the properties of the wild-type channels. Here we show that hCx31 channels, like other connexin channels, are gated by voltage and close at low pH and when exposed to long-chain alkanols. Single-channel conductance of the fully open channel is approximately 85 pS, and it is permeable to Lucifer yellow, Alexa Fluor(350), ethidium bromide, and DAPI, which have valences of -2, -1, +1, and +2, respectively. In contrast to what has been reported for mouse Cx31, hCx31 appears to form functional heterotypic channels with all four connexins tested, Cx26, Cx30, Cx32, and Cx45. These findings provide an important first step in evaluating the pathogenesis of inherited human diseases associated with mutations in the gene for Cx31.