The distribution of microfilament bundles, intermediate filaments, microtubules, vinculin plaques, and extracellular fibronectin matrices was examined by indirect immunofluorescence in X-ray-transformed C3H/10T½ cells. The transformed phenotype correlated with a lack of actin cables and vinculin-containing plaques. Prolonged treatment of these transformed cells with interferon resulted in a reversion of the transformed phenotype which was associated with the reappearance of extensive microfilament bundles together with vinculin-containing plaques. Both interferon-treated and untreated transformed cells displayed a similar distribution of mitochondria, intermediate filaments, microtubules, and fibronectin. Significant qualitative and quantitative differences between the two types of cells were found in the proteins associated with the cytoskeletal framework by two-dimensional gel analysis.