An extended synthetic oligonucleotide (58-mer) has been used to identify and characterize a human liver gap junction cDNA. The cDNA is 1,574 bases long and contains the entire coding region for a gap junction protein. In vitro translation of the RNA products of this cDNA is consistent with it coding for a 32,022-D protein. Southern blot analysis indicates that the gap junction gene is present as a single copy, and that it can be detected in a variety of organisms using the human liver cDNA as a probe. The human cDNA has been used to screen a rat liver cDNA library, and a rat liver junction cDNA clone has been isolated. The rat liver clone is 1,127 bases in length, and it has strong sequence homology to the human cDNA in the protein-coding region, but less extensive homology in the 3'-untranslated region.