Several laboratories have demonstrated a decrease in gap junctional communication in cells transformed by the src oncogene of the Rous sarcoma virus. The decrease in gap junctional communication was associated with tyrosine phosphorylation of the gap junction protein, connexin 43 (Cx43). This study was initiated to determine if the phosphorylation of Cx43 is the result of a direct kinase-substrate interaction between the highly active tyrosine kinase, pp60v-src, and Cx43. Previous biochemical studies have been limited by the low levels of Cx43 protein in fibroblast cell lines. To obtain larger quantities of Cx43, we constructed a recombinant baculovirus expressing Cx43 in Spodoptera frugiperda (Sf-9) cells and subsequently purified the expressed Cx43 by immunoaffinity chromatography. We observed that this partially purified Cx43 was phosphorylated on tyrosine in vitro in the presence of kinase-active pp60src. Phosphotryptic peptide mapping indicated that the in vitro phosphorylated Cx43 contained phosphopeptides which comigrated with a subset of tryptic peptides prepared from Cx43 phosphorylated in vivo. Furthermore, coinfection of Sf-9 cells with recombinant baculoviruses encoding pp60v-src and Cx43 resulted in the accumulation of phosphotyrosine in Cx43. Taken together, the evidence presented in this paper demonstrates that kinase active pp60c-src is capable of phosphorylating Cx43 in a direct manner. Since the presence of phosphotyrosine on Cx43 is correlated with the down-regulation of gap-junctional communication, these results suggest that pp60v-src regulates gap junctional gating activity via tyrosine phosphorylation of Cx43.