NIH 3T3 fibroblasts stably transformed with a constitutively active isoform of p21^Ras, H-Ras^V12 (v-H-Ras or EJ-Ras), produced large amounts of the reactive oxygen species superoxide (·O₂⁻). ·O₂⁻ production was suppressed by the expression of dominant negative isoforms of Ras or Rac1, as well as by treatment with a farnesyltransferase inhibitor or with diphenylene iodonium, a flavoprotein inhibitor. The mitogenic activity of cells expressing H-Ras^V12 was inhibited by treatment with the chemical antioxidant N-acetyl-L-cysteine. Mitogen-activated protein kinase (MAPK) activity was decreased and c-Jun N-terminal kinase (JNK) was not activated in H-Ras^V12-transformed cells. Thus, H-Ras^V12-induced transformation can lead to the production of ·O₂⁻ through one or more pathways involving a flavoprotein and Rac1. The implication of a reactive oxygen species, probably ·O₂⁻, as a mediator of Ras-induced cell cycle progression independent of MAPK and JNK suggests a possible mechanism for the effects of antioxidants against Ras-induced cellular transformation.