Cell-substrate adherence in cultures of chicken embryo fibroblasts was examined by determining the number of cells which could be detached from the culture dish by a stream of medium. Transformed cells were significantly less adherent than their normal counterparts. In cultures infected with a mutant of Rous sarcoma virus which is temperature-conditional for transformation, adherence changed promptly following a temperature shift. This change did not require progression through the cell cycle. The transformation-specific decrease in adherence required new protein synthesis, but the restoration of adherence which occurred following a shift to the restrictive temperature could occur in the absence of new protein synthesis. Inhibitor experiments suggested the importance of microfilaments and perhaps microtubules in the changes in detachability. In addition, there was a positive correlation between levels of surface LETS protein and cell substrate adherence following a temperature shift, although it seems probable that the bulk of the surface LETS is neither necessary nor sufficient for maintenance of normal cell substrate adherence.