We have isolated and characterized a human genomic clone containing the complete coding region of connexin31 (Cx31). Similar to rodent Cx31, the coding region of human Cx31 is completely contained within the second exon and consists of 810 nucleotides. The deduced human Cx31 polypeptide consists of 270 amino acids with a predicted molecular mass of 30.818 kDa. Its sequence is most similar to mouse Cx31 (82.6% identical amino acids) and rat (83.0% identical amino acids), but shows considerably fewer potential sites of phosphorylation. After Northern blot hybridization, two Cx31 transcripts of 2.2 and 1.8 kb were detected in total RNA of the human keratinocyte cell line HaCaT and two transcripts of 2.2 and 1.9 kb in total RNA of E6/E7 transfected human keratinocytes (HEK cells). Using affinity-purified rabbit antibodies to mouse Cx31, immunofluorescence analysis demonstrated relatively weak expression of human Cx31 in HaCaT and HEK cells. The Cx31 gene exists as a single copy gene in the human genome and was mapped to the chromosomal region 1p34-p36 by analyzing human-mouse somatic cell hybrids.