Gap junctions are widely distributed structures that mediate communication between cells. The channels that allow passage of small molecules between adjacent cells are made up of oligomeric proteins (connexins) that are encoded by a family of related genes. By probing somatic cell hybrid DNA on Southern filters with rat or human cDNAs or human genomic fragments, we have mapped four functioning gap junction genes, (alpha 1, beta 1, beta 2, and alpha 3), to different sites on human chromosomes: GJA1 (connexin43) to 6p21.1-q24.1; GJB1 (connexin32) to Xcen-q22; GJB2 (connexin26) to 13; and GJA3 (connexin46) also to 13, probably near GJB2. The GJA3 probe also hybridized to a restriction fragment that was mapped to chromosome 1. A GJA1-related pseudogene GJA1P was assigned to chromosome 5. The homologous loci in mouse were assigned to regions of known conserved syntenic groups: Gja-1 to chromosome 10; Gjb-1 to XD-F4 and Gjb-2 to 14. Of two sites of hybridization with the GJA3 probe, on mouse 14 and 5, we assume that the site on 14 corresponds to the GJA3 locus on human 13. Based on these data, additional members of this family of related genes can be isolated and characterized, and possible human and mouse mutations can be identified.