The pp60v-src tyrosine kinase disrupts gap junctional communication in transformed fibroblasts and induces the phosphorylation of the gap junction protein, connexin43, on tyrosine. We report here that the p130gag-fps tyrosine kinase also profoundly disrupted gap junctional communication and markedly increased the phosphorylation of connexin43 which appeared to result from an accumulation of phosphotyrosine and phosphoserine. The disruption of gap junctional communication by pp60v-src and p130gag-fps did not appear to result from the gross alteration of gap junction plaques. Furthermore, two-dimensional phosphotryptic peptide mapping showed that the v-Src and V-Fps kinases stimulated the phosphorylation of multiple connexin43 peptides which contained phosphotyrosine and/or phosphoserine. Phosphotyrosine was detected in two connexin43 phosphotryptic peptides from v-src-tranformed cells which suggested that more than one connexin43 tyrosine site may be recognized by pp60v-src in fibroblasts. The apparent higher levels of phosphoserine-containing connexin43 peptides in the oncogene-transformed cells pointed to the possibility that pp60v-src and p130gag-fps may also modulate connexin43 function through mechanism(s) involving the activation of signaling serine kinases. Taken together, these results suggested that connexin43 is a common target of the v-Src and v-Fps tyrosine kinase oncoproteins.