We have developed a generalized approach, using two hybrid interactions, to isolate Ha-Ras effector loop mutations that separate the ability of Ha-Ras to interact with different downstream effectors. These mutations attenuate or eliminate Ha-ras (G12V) transformation of mammalian cells, but retain complementary activity, as demonstrated by synergistic induction of foci of growth-transformed cells, and by the ability to activate different downstream components. The transformation defect of Ha-ras (G12V, E37G) is rescued by a mutant, rafl, that restores interaction. These results indicate that multiple cellular components, including Rafl, are activated by Ha-Ras and contribute to Ha-Rasinduced mammalian cell transformation.