Cell shape is involved in a variety of cellular activities including proliferation, adhesion, migration, and transformation. Agents known to promote differentiation, such as retinoic acid, butyrate, and dibutyryl cyclic AMP, induce marked alterations in cell shape which are often accompanied by changes in cell functions. In this paper we study the effects of the differentiating polar solvent dimethyl sulfoxide (DMSO) on cytoskeleton, adhesion, and growth properties of cultured mouse B16 melanoma cells. DMSO induced a progressive reorganization of the cytoskeleton which was fully developed in 4 days of continuous exposure to the agent. DMSO-treated cells developed thick and regularly oriented microfilament bundles of the stress fiber type ending at vinculin-rich areas of focal contact between the ventral membrane and the substratum (interference reflection microscopy-dark adhesion plaques). Such a rearrangement of the cytoskeleton resulted in increased adhesion to the substratum and inhibition of cell growth in comparison to control untreated cells. Cells which became highly flattened and tightly adherent after exposure to DMSO for 4 days progressively reverted their phenotype to that of control untreated cells within 3 days of DMSO withdrawal. Namely, they lost stress fibers and adhesion plaques, became rounded and less adherent, and increased their growth rate. These results indicate that DMSO can change the transformed appearance of B16 mouse melanoma cells to a phenotype which is typical of a variety of nontransformed cells in culture.