Single site mutations in connexins have provided insights about the influence specific amino acids have on gap junction synthesis, assembly, trafficking and functionality. We have discovered a single point mutation that eliminates functionality without interfering with gap junction formation. The mutation occurs at a threonine residue located near cytoplasmic end of the third transmembrane helix. This threonine is strictly conserved among members of the and connexin sub-groups, but not the sub-group. In HeLa cells, connexin43 and connexin26 mutants are synthesized, traffic to the plasma membrane and make gap junctions with the same overall appearance as wild type. We have isolated connexin26T135A gap junctions both from HeLa cells and baculovirus infected insect Sf9 cells. Using cryo-electron microscopy and correlation averaging, difference images revealed a small but significant size change within the pore region and a slight rearrangement of the subunits between mutant and wild-type connexons expressed in Sf9 cells. Purified, detergent solubilized mutant connexons contain both hexameric and partially disassembled structures while wild-type connexons are almost all hexameric, suggesting that the 3D mutant connexon is unstable. Mammalian cells expressing gap junction plaques composed of either connexin43T154A or connexin26T135A showed an absence of dye coupling. When expressed in Xenopus oocytes, these mutants, as well as a cysteine substitution mutant of connexin50 (connexin50T157C) failed to produce electrical coupling in homotypic and heteromeric pairings with wild type in a dominant-negative effect. This mutant may be useful as a tool for knocking-down or knocking-out connexin function in vitro or in vivo.