We have previously demonstrated that epidermal growth factor induced a rapid, transient decrease in gap junctional communication and increase in serine phosphorylation on the connexin-43 gap junction protein in T51B rat liver epithelial cells. The kinase(s) responsible for phosphorylation and specific serine targets in connexin-43 have not been identified. There are three consensus mitogen-activated protein (MAP) kinase serine phosphorylation sequences in the carboxyl-terminal tail of connexin-43 and purified MAP kinase phosphorylated connexin-43 in vitro on tryptic peptides that comigrated with a subset of peptides from connexin-43 phosphorylated in vivo in cells treated with epidermal growth factor. These data suggested that MAP kinase may phosphorylate connexin-43 directly in vivo. We have utilized a glutathione S-transferase fusion protein containing the cytoplasmic tail of connexin-43 to characterize MAP kinase phosphorylation. Site-directed mutagenesis, phosphotryptic peptide analysis, and peptide sequencing have confirmed that MAP kinase can phosphorylate connexin-43 at Ser255, Ser279, and Ser282, which correspond to the consensus sites recognized earlier. Characterization of MAP kinase-mediated phosphorylation of connexin-43 has defined potential targets for phosphorylation in vivo following activation of the epidermal growth factor receptor and has provided the basis for studies of the effects of phosphorylation, at specific molecular sites, on the regulation of gap junctional communication.