The functional state of gap junctions and the state of phosphorylation of connexin43 (Cx43), the major gap junction protein in rat heart, were evaluated in primary cultures of neonatal rat cardiocytes. Functional coupling was greatly reduced after treatment with staurosporine (ST), a protein kinase inhibitor. The ST-induced reduction in cell coupling was reversed by activation of protein kinase C (PKC) with 12-O-tetradecanoylphorbol 13-acetate (TPA). The cellular distribution of Cx43, as detected by immunofluorescence, was not grossly affected by either ST alone or ST plus TPA. Although immunoblot analysis did not detect significant changes in the relative amounts of the unphosphorylated and individual phosphorylated forms of Cx43 after each treatment, the level of 32P-incorporation into Cx43 of radiolabeled cells was significantly affected. Consistent with their known properties, treatment with ST reduced, and combined treatment with TPA and ST increased, the level of 32P-incorporation into Cx43. Two-dimensional tryptic phosphopeptide maps of 32P-labeled Cx43 indicated that a distinct subset of the phosphopeptides that are present under basal conditions were affected by ST or ST/TPA treatments, with TPA-induced phosphorylation occurring at the ST-sensitive sites. However, the ST/TPA-sensitive tryptic phosphopeptides did not comigrate with others that were derived from in vitro phosphorylation by PKC of a recombinant C-terminal Cx43 peptide (Cx43[243-382]). Although a PKC-dependent mechanism appears to be involved in the regulation of functional coupling between neonatal rat cardiocytes, PKC itself may not be the final mediator of Cx43 phosphorylation.