We have investigated the localization and phosphorylation of the fibronectin receptor in chick embryo fibroblasts transformed either by wild-type Prague C strain Rous sarcoma virus, which induces a rounded, less adhesive phenotype with a loss of surface fibronectin and gross cytoskeletal changes, or transformed by rASV2234.3, a variant which induces a flat, adhesive morphology with the retention of surface fibronectin and more normal cytoskeleton. Immunofluorescence showed co-distribution of pp60v-src with integrin and fibronectin fibrils in rASV2234.3-transformed cells. Total levels and surface expression of integrin were unchanged in both transformed cell types compared with untransformed cells. However, whereas integrin band 3 (beta 1 subunit) in Prague C-transformed cells was hyperphosphorylated on tyrosine, this was reduced virtually to normal in rASV2234.3-transformed cells. A similar differential phosphorylation of integrin band 3 could be found in membranes phosphorylated in vitro. rASV2234.3 pp60v-src was less efficient in phosphorylating a synthetic peptide containing the putative integrin tyrosine phosphorylation site, indicating that this variant pp60v-src has an altered substrate specificity compared to Prague C pp60v-src. The correlation between tyrosine-specific phosphorylation of integrin and loss of surface fibronectin suggests that this could play an important role in the induction of the transformed phenotype.