The protein kinase KSR-1 is a recently identified participant in the Ras signaling pathway. The subcellular localization of KSR-1 is variable. In serum-deprived cultured cells, KSR-1 is primarily found in the cytoplasm; in serum-stimulated cells, a significant portion of KSR-1 is found at the plasma membrane. To identify the mechanism that mediates KSR-1 translocation, we performed a yeast two-hybrid screen. Three clones that interacted with KSR-1 were found to encode the full-length γ10 subunit of heterotrimeric G- proteins. KSR-1 also interacted with γ2 and γ3 in a two-hybrid assay. Deletion analysis demonstrated that the isolated CA3 domain of KSR-1, which contains a cysteine-rich zinc finger-like domain, interacted with γ subunits. Coimmunoprecipitation experiments demonstrated that KSR-1 bound to β1γ3 subunits when all three were transfected into cultured cells. Lysophosphatidic acid treatment of cells induced KSR-1 translocation to the plasma membrane from the cytoplasm that was blocked by administration of pertussis toxin but not by dominant-negative Ras. Finally, transfection of wild-type KSR-1 inhibited β1γ3-induced mitogen-activated protein kinase activation in cultured cells. These results demonstrate that KSR-1 translocation to the plasma membrane is mediated, at least in part, by an interaction with βγ and that this interaction may modulate mitogen- activated protein kinase signaling.