TPA is known to cooperate with an activated Ras oncogene in the transformation of rodent fibroblasts, but the biochemical mechanisms responsible for this effect have not been established. In the present study we used c-fos promoter-luciferase constructs as reporters, in transient transfection assays, in NIH3T3 cells to assess the mechanism of this cooperation. We found a marked synergistic interaction between TPA and a transfected v-Ha-ras oncogene in the activation of c-fos promoter and SRE. SRE has binding sites for TCF and SRF. A dominant-negative Ras (ras-N17) inhibited the TPA-Ras synergy by blocking the PKC-MAPK-TCF pathway. Dominant-negative RhoA and Rac1 (but not Cdc42Hs) inhibited the TPA-Ras synergy by blocking the Ras-Rho-SRF signaling pathway. Constitutively active PKCalpha and PKCepsilon showed synergy with v-Ras. These results suggest that the activation of two distinct pathways such as Ras-Raf-ERK-TCF pathway and Rho-SRF pathway are responsible for the induction of c-fos by TPA and Ras in mitogenic signaling pathways.