We used the yeast two-hybrid system to identify proteins that interact directly with Gα(o). Mutant-activated Gα(o) was used as the bait to screen a cDNA library from chick dorsal root ganglion neurons. We found that Gα(o) interacted with several proteins including Gz-GTPase-activating protein (Gz- GAP), a new RGS protein (RGS17), a novel protein of unknown function (IP6), and Rap1GAP. This study focuses on Rap1GAP, which selectively interacts with Gα(o) and Gα(i) but not with Gα(s) or Gα(q). Rap1GAP interacts more avidly with the unactivated Gα(o) as compared with the mutant (Q205L)- activated Gα(o). When expressed in HEK-293 cells, unactivated Gα(o) co- immunoprecipitates with the Rap1GAP. Expression of chick Rap1GAP in PC-12 cells inhibited activation of Rap1 by forskolin. When unactivated Gα(o) was expressed, the amount of activated Rap1 was greatly increased. This effect was not observed with the Q205L-Gα(o). Expression of unactivated Gα(o) stimulated MAP-kinase (MAPK1/2) activity in a Rap1GAP-dependent manner. These results identify a novel function of Gα(o), which in its resting state can sequester Rap1GAP thereby regulating Rap1 activity and consequently gating signal flow from Rap1 to MAPK1/2. Thus, activation of GO could modulate the Rap1 effects on a variety of cellular functions.