Connexin43 phosphorylation was analysed in non-transformed and pp60v-src-transformed Rat-1 fibroblasts. Connexin43 appeared to be the primary connexin expressed in these cells. Although gap-junctional communication was disrupted in pp60v-src-transformed cells, they contained more connexin43 protein and RNA than their non-transformed counterpart. Connexin43 was phosphorylated within minutes of its synthesis in both cell types and appeared to be degraded while in the phosphorylated state. Phosphopeptide and phosphoamino acid analyses suggested that connexin43 in both cell types contained at least five fragments with serine phosphorylation. The major difference in connexin43 phosphorylation between the pp60v-src-transformed and non-transformed cells was that, whereas approx. 70% of the phosphorylated connexin43 in the former contained phosphotyrosine, this phosphoamino acid was not detected in connexin43 isolated from the latter cells. These data support the hypothesis that phosphorylation of connexin43 on tyrosine is critical for the blockade of gap-junctional communication which occurs concomitantly with transformation by the pp60v-src oncogene.