The effects of scatter factor on the cytoskeleton of MDCK and PtK2 cells are described. During the first 6 h after the addition of scatter factor, MDCK cells were found to increase their projected areas twofold, as well as the number and size of their F-actin stress fibers. In contrast PtK2 cells showed no change in their projected areas or in their stress fiber content. However, when both MDCK and PtK2 cells began to separate and scatter after approximately 6 h, the size and number of stress fibers was found to decrease considerably. Unscattered PtK2 cells and cells treated with scatter factor which had yet to scatter showed focal contacts present over the whole ventral surface, as judged by staining for both vinculin and talin. After treated cells separated, both vinculin and talin staining were mainly present in focal contacts on the ventral surfaces of the cell bodies and the distal ends of the processes. However, the cell processes showed few focal contacts along their lengths. The distribution of microtubules and vimentin and keratin intermediate filaments also did not change significantly until scattering had occurred. After cell separation, the processes were always packed with microtubules which were often, but not always, rich in detyrosinated alpha-tubulin and often, but not always, packed with intermediate filaments. All these changes in cytoskeletal organization are consistent with the adoption of a much more motile phenotype. The changes found are compared with those brought about by transformation.