Mutations in Ras GTPase and various other components of the Ras signaling pathways are among the most common genetic alterations in human cancers and also have been identified in several familial developmental syndromes. Over the past few decades it has become clear that the activity or the oncogenic potential of Ras is dependent on the nonreceptor tyrosine kinase Src to promote the Ras/Raf/MAPK pathway essential for proliferation, differentiation, and survival of eukaryotic cells. However, no direct relationship between Ras and Src has been established. We show here that Src binds to and phosphorylates GTP-, but not GDP-, loaded Ras on a conserved Y32 residue within the switch I region in vitro and that in vivo, Ras-Y32 phosphorylation markedly reduces the binding to effector Raf and concomitantly increases binding to GTPase-activating proteins and the rate of GTP hydrolysis. These results suggest that, in the context of predetermined crystallographic structures, Ras-Y32 serves as an Src-dependent keystone regulatory residue that modulates Ras GTPase activity and ensures unidirectionality to the Ras GTPase cycle.