It is known that in Clone 9 (C9) cells, intercellular gap junctional communication (IGJC) is rapidly blocked by the tumor promoter phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), but it recovers spontaneously a few hours later and becomes refractory to TPA (Yada et al., J. Membr. Biol. 88, 217-232 (1985)). We now report that gap junctions between C9 cells contain at least two junctional proteins, connexin26 (Cx26) and connexin43 (Cx43), and that the TPA-induced changes in IGJC correlate temporally to changes in the state of phosphorylation of Cx43. The latter changes were prevented by inhibition of protein kinase C. Phosphoamino acid analysis and two-dimensional tryptic peptide maps of 32P-labeled Cx43 showed that during the TPA-induced phosphorylation at least two of the phosphorylated forms of Cx43 were differentially phosphorylated in seryl residues as compared to control. TPA induced a drastic reduction in junctional conductance as well as a redistribution of unitary gap junction channel event sizes seen in control cells. These changes were associated with retrieval of Cxs from the plasma membrane. Reappearance of gap junctions formed by Cx43 but not by Cx26 accounted for the spontaneous recovery in IGJC. It is proposed that gap junctions between C9 cells contain two types of channels each formed by Cx43 or Cx26 and that they are differentially affected during the action of TPA.