Mitogenic stimulation of cardiomyocytes is associated with decreased gap junction coupling and protein kinase C (PKC)-mediated phosphorylation of the gap junction protein connexin43 (Cx43). Identification of and interference with the amino acid(s) that becomes phosphorylated in response to stimulation are important steps towards defining the relationship between Cx43 phosphorylation and cell cycle. Using immunoblotting and phosphospecific antibodies we were able to show that serine-262 (S262) on Cx43 becomes phosphorylated in response to growth factor or PKC stimulation of cardiomyocytes. To examine the effect of Cx43, S262 phosphorylation and cell-cell contact (and/or coupling) on DNA synthesis, we overexpressed wild-type (wt) or mutant Cx43, carrying a S262-to-alanine (S262A, simulating the unphosphorylated state) or a S262-to-aspartate (S262D, simulating constitutive phosphorylation) substitutions in cultures of cell-cell contact forming or isolated cardiomyocytes. Overexpression of wt-Cx43 caused a significant decrease in DNA synthesis irrespective of the presence of cell-cell contact. In cell-cell contact forming cultures, the S262D mutation reversed while the S262A mutation increased the inhibitory effect of Cx43. In the absence of cell-cell contact, the S262-Cx43 mutations had no significant effect on Cx43 inhibition of DNA synthesis. Dye-coupling, evaluated by scrape-loading, indicated increased gap junction permeability in S262A (compared to wt or S262D) overexpressing myocytes. We conclude that Cx43 inhibits cardiomyocyte DNA synthesis irrespectively of cell-cell contact or coupling. Cell-cell contact, and possibly gap junction-mediated communication is required, however, in order to reverse Cx43 inhibition of DNA synthesis by S262 phosphorylation.