The gap junction channel mediates an important form of intercellular communication, but its detailed study is hindered by inaccessibility in situ. We show here that connexin32, the major protein composing junctional channels in rat liver, forms ion channels in single bilayer membranes. The properties of these reconstituted connexin32 channels are characterized and compared with those of gap junction channels. The demonstration that connexin32 forms channels in single membranes has implications for assembly and regulation of junctional channels, and permits detailed study of the gating, permeability and modulation of this channel-forming protein.