Control mechanism of gap junctional permeability by phosphorylation was examined in a model system in which gap junctional hemichannels were reconstituted in lipid vesicles. Connexin 43 (Cx43) and Connexin 32 (Cx32) were immunoaffinity-purified from rat brain and liver respectively and were reconstituted into unilamellar phospholipid liposomes. The activity of reconstituted channels was measured by monitoring the permeability of liposomes containing connexins. The liposomes containing the connexin proteins were fractionated on the basis of permeability to urea/sucrose in an iso-osmolar density gradient and the sucrose-permeable and impermeable liposomes were separated in the gradient. The liposomes permeable to sucrose were also permeable to a communicating dye molecule, Lucifer Yellow. It was found that the channel activities of the reconstituted connexin hemichannels were pH-sensitive and directly dependent on the phosphorylation states of connexin proteins. The permeability of liposomes containing Cx43 channels was increased by treating the liposomes with calf intestinal phosphatase (CIP). Moreover liposomes formed with Cx43 which was dephosphorylated by CIP treatment revealed increased permeability to sucrose. The permeability of liposomes containing Cx32 channels was decreased by treating the connexins contained in liposomes with protein kinase C and protein kinase A, and the changes of permeability were reversed by dephosphorylating connexin by CIP treatment. The permeability of liposomes containing Cx43 was also decreased by phosphorylating the connexin by MAP kinase treatment. Data provide evidence that the gap junctional permeability is directly dependent on the phosphorylation of gap junction proteins.