Chicken embryo fibroblasts transformed with the Ta and ts68 mutants of Rous Sarcoma virus (RSV) were enucleated and studied for their capacity to express reversibly the transformed phenotype in response to temperature changes. After shift to the permissive temperature (35 degrees C), the cytoplasts acquired a transformed morphology and displayed characteristic ruffles and microvilli at their surface. As detected by immunofluorescence, they also lost their actin filament cables and exhibited characteristic changes in the pattern of cell surface structures containing LETS protein. Expression of all these transformation parameters was reversible after shiftback to the nonpermissive temperature (41 degrees C). These results indicate that a whole set of changes characteristic for the transformed phenotype can be expressed independently of the cell nucleus. In contrast, ts mutant-infected cytoplasts were no longer able to respond to temperature shifts with changes in their hexose transport rate. Cytoplasts prepared from cells grown at 41 degrees C retained their low rate of hexose uptake after shift to 35 degrees C, whereas cytoplasts from cells grown at 35 degrees C exhibited a high rate of hexose transport even after 10 hr of shift to 41 degrees C. These results are in accordance with the hypothesis that the product of the src gene of RSV represents a multifunctional protein which acts independently on nuclear and extranuclear sites.