Antisera were raised against a synthetic peptide (src-c) containing the six COOH-terminal amino acids of p60src, the transforming protein of Rous sarcoma virus (RSV). Antibodies specific for the src-c peptide were purified by affinity chromatography and then used to study the location of p60src in transformed cells. The distribution of p60src was compared to that of vinculin, a candidate cytoskeletal substrate of p60src, by indirect double immunofluorescence microscopy. In RSV-transformed rat, mouse, and chicken cells, an extensive codistribution of p60src with vinculin was observed. Both proteins were concentrated in the few remaining focal adhesion plaques, in transformation-induced rosette clusters at the ventral cell surface, and in cell-cell contact areas. In addition, antibodies to both proteins stained the cytoplasm diffusely. In all cells examined, the immunofluorescent staining patterns produced by antibodies to the src-c peptide were indistinguishable from those obtained by immunolabeling of p60src with sera from RSV-infected tumor-bearing rabbits. The excellent agreement of the results obtained with two completely independent antibody preparations indicates strongly that the observed immunolabeling patterns correctly define the intracellular distribution of p60src. The significance of the intracellular location of p60src to the transforming activities of the protein is discussed.