A vector expressing a dominant negative mutant of Raf-1 was stably introduced into BALB/c 3T3 cells expressing a temperature-sensitive derivative of v-Src. High levels of the Raf-mutant were detected in these cells. Expression of the Raf-1 mutant blocked v-Src-induced transformation, as determined by reversion to a flat non-transformed morphology and the inability to form colonies in soft agar. Cells transfected with the parental vector lacking the mutant Raf-1 could be transformed by v-Src. These data suggest that intracellular signals activated by v-Src that are mediated by Raf-1 are required for transformation by v-Src.