Human HeLa or SkHep1 cells, defective in intercellular communication through gap junctions, were transfected with coding sequences of murine connexin40 (Cx40) and -43. The transfected cells were restored in gap junctional coupling as shown by 100-fold increased electrical conductance. When studied by the double whole-cell patch-clamp technique, Cx40 HeLa transfectants exhibited single channel conductances of gamma = 121 +/- 7 pS and gamma = 153 +/- 5 pS. They were voltage gated with an equivalent gating charge of z = 4.0 +/- 0.5 for a voltage of half-maximal inactivation U0 = 44 +/- 7 mV. The corresponding values of connexin43 (Cx43) HeLa transfectants are: gamma = 60 +/- 4 pS and gamma = 40 +/- 2 pS as well as z = 3.7 +/- 0.8 and U0 = 73 +/- 7 mV. Transfer of the dye Lucifer Yellow was always considerably lower in Cx40- than in Cx43-transfectants though their total junctional conductance was similar or even higher than for Cx43-transfectants. In order to characterize cell and tissue distribution as well as phosphorylation of connexin40 and -43 proteins, antibodies to C-terminal oligopeptides of these proteins were prepared and used for immunoblotting, immunoprecipitation, and immunofluorescence analysis of transfected cells where they exhibited the punctate pattern characteristic of gap junctions on contacting membranes. Phosphorylation of connexin40 was shown by immunoprecipitation from 32P-labeled, transfected SkHep1 cells. Analyses of protein distribution in tissues revealed that the amount of connexin40 detected in heart was higher than in lung which is the inverse of the level of connexin40 mRNA in these tissues, suggesting posttranscriptional control of expression. Connexin40 protein in adult mouse heart and skin is about 20-fold more abundant than in the corresponding embryonic tissue. Connexin43 in adult mouse heart appears to be more highly phosphorylated than in embryonic heart or in transfected human cells.