Most gap junction channels are sensitive to the voltage difference between the two cellular interiors, termed the transjunctional voltage (Vj). In several junctions, the conductance transitions induced by Vj show more than one kinetic component. To elucidate the structural basis of the fast and slow components that characterize the Vjdependence of connexin-32 (Cx32) and connexin-43 (Cx43) junctions, we created deletions of both connexins, where most of the carboxy-terminal (CT) domain was removed. The wild-type and “tailless” mutants were expressed in paired Xenopus oocytes, and the macroscopic gating properties were analyzed using the dual voltage clamp technique. Truncation of the CT domain of Cx32 and Cx43 abolished the fast mechanism of conductance transitions and induced novel gating properties largely attributable to the slow mechanism of gating. The formation of hybrid junctions comprising wild-type and truncated hemichannels allowed us to infer that the fast and slow components of gating reside in each hemichannel and that both gates close at a negative Vj on the cytoplasmic side. Thus we conclude that the two kinetic components of Vj-sensitive conductance are a result of the action of two different gating mechanisms. They constitute separate structures in the Cx32 and Cx43 molecules, the CT domain being an integral part of fast Vj gating.