The molecular mechanisms underlying the voltage dependence of intercellular channels formed by the family of vertebrate gap junction proteins (connexins) are unknown. All vertebrate gap junctions are sensitive to the voltage difference between the cells, defined as the transjunctional voltage, Vj (refs 1, 2), and most appear to gate by the separate actions of their component hemichannels. The heterotypic Cx32/Cx26 junction displays an unpredicted rectification that was reported to represent a novel Vj dependence created by hemichannel interactions, mediated in part by the first extracellular loop E1 (ref. 9). Here we show that aspects of the rectification of Cx32/Cx26 junctions are explained by opposite gating polarities of the component hemichannels, and that the opposite gating polarity of Cx32 and Cx26 results from a charge difference in a single amino-acid residue located at the second position in the N terminus. We also show that charge substitutions at the border of the first transmembrane (M1) and E1 domains can reverse gating polarity and suppress the effects of a charge substitution at the N terminus. We conclude that the combined actions of residues at the N terminus and M1/E1 border form a charge complex that is probably an integral part of the connexin voltage sensor. A consistent correlation between charge substitution and gating polarity indicates that Cx26 and Cx32 voltage sensors are oppositely charged and that both move towards the cytoplasm upon hemichannel closure.