— Two gap junction proteins, connexin43 (Cx43) and connexin45 (Cx45), are coexpressed in many cardiac and other cells. Homomeric channels formed by these proteins differ in unitary conductance, permeability, and regulation. We sought to determine the ability of Cx43 and Cx45 to oligomerize with each other to form heteromeric gap junction channels and to determine the functional and regulatory properties of these heteromeric channels. HeLa cells were transfected with Cx45 or (His)₆-tagged Cx43 or sequentially transfected with both connexins. Immunoblots verified production of the transfected connexins, and immunofluorescence demonstrated that they were colocalized in the HeLa-Cx43(His)₆/Cx45 cells. Connexons were solubilized from HeLa-Cx43(His)₆/Cx45 cells by using Triton X-100 and were applied to a Ni²⁺-NTA column, which binds the His₆ sequence. Cx45 was coeluted from the column with Cx43(His)₆, demonstrating that some hemichannels contain both connexins. Single-channel recordings showed that the HeLa-Cx43(His)₆/Cx45 cells exhibited single-channel conductances that were not observed in cells expressing either connexin alone. Dye-coupling experiments showed that HeLa-Cx43(His)₆ cells readily passed Lucifer yellow and N-(2-aminoethyl)biotinamide hydrochloride (neurobiotin); in contrast, HeLa-Cx45 and HeLa-Cx43(His)₆/Cx45 cells showed extensive intercellular passage of neurobiotin but little coupling with Lucifer yellow. Treatment with the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate reduced junctional conductance in cells expressing Cx43, Cx45, or both connexins, but it reduced the extent of neurobiotin transfer only in HeLa-Cx43(His)₆ and HeLa-Cx43(His)₆/Cx45 cells but not in the HeLa-Cx45 cells. Thus, biochemical and electrophysiological evidence suggests that Cx43 and Cx45 extensively mix to form heteromeric channels; however, individual connexin components dominate aspects of the physiological behavior of these channels.