Oncogenic H-Ras G12V and its variants have been shown to inhibit muscle differentiation. However, the role of proto-oncogenic Ras (c-Ras) in muscle differentiation remains unclear. The active GTP-bound form of Ras has been known to associate with diverse effectors including Raf, phosphatidylinositol 3-kinase (PI3K), Ral-GDS, and other molecules to transmit downstream signals. We hypothesize that c-Ras may stimulate muscle differentiation by selectively activating PI3K, an important mediator for muscle differentiation. In our experiments, inhibition of c-Ras by farnesyltransferase inhibitors and a dominant negative form of H-Ras (Ras S17N) suppressed muscle differentiation. Consistently, individual knockdown of H-Ras, K-Ras, and N-Ras by siRNAs all blocked muscle differentiation. Interestingly, we found that c-Ras preferentially interacts with PI3K rather than its major binding partner c-Raf, during myogenic differentiation, with total c-Ras activity remaining unchanged. PI3K and its downstream myogenic pathway, the Nox2/NF-kappaB/inducible nitric oxide synthase (iNOS) pathway, were found to be suppressed by inhibition of c-Ras activity during differentiation. Furthermore, expression of a constitutively active form of PI3K completely rescued the differentiation block and reactivated the Nox2/NF-kappaB/iNOS pathway in c-Ras-inhibited cells. On the basis of our results, we conclude that contrary to oncogenic Ras, proto-oncogenic H-Ras, K-Ras, and N-Ras are directly involved in the promotion of muscle differentiation via PI3K and its downstream signaling pathways. In addition, PI3K pathway activation is associated with a concurrent suppression of the otherwise predominantly activated Raf/Mek/Erk pathway.