Smooth muscle alpha-actin promoter is repressed in ras-transformed fibroblast cells and derepressed in revertant cells. We have recently shown that serum response factor (SRF) which can bind to the serum response elements (SREs) present in the alpha-actin promoter, can activate alpha-actin promoter activity in ras-transformed cells and suppress transformation by ras. Agents that stimulate SRF expression and alpha-actin promoter activity in ras-transformed cells are expected to be potential candidates as antitumor agents. In this study, we show that treatment of ras-transformed cells with antitumor agents such as taxol, vincristine, vinblastine, colchicine, and nocodazole leads to 5- to 7-fold activation of alpha-actin promoter driven CAT activity, whereas there was very little effect on thymidine kinase promoter driven CAT activity. This activation occurred at subcytotoxic concentrations of these agents and correlated with inhibition of cell cycle progression. Furthermore, these agents stimulated SRF expression in ras-transformed cells, as measured by its SRE binding activity. The increase in alpha-actin expression is accompanied by the restoration of actin filaments into organized bundles. These results suggest a novel mechanism by which antimitotic agents suppress the ras-transformed phenotype.