We have generated stable IIC9 cell lines, Goa1 and Goa2, that overexpress full-length antisense G(o)α RNA. As shown previously, expression of antisense G(o)α RNA ablated the a subunit of the heterotrimeric G protein, G(o), resulting in growth in the absence of mitogen. To better understand this change in IIC9 phenotype, we have characterized the signaling pathway and cell cycle events previously shown to be important in control of IIC9 G1/S phase progression. In this paper we clearly demonstrate that ablation of G(o)α results in growth, constitutively active Ras/ERK, elevated expression of cyclin D1, and constitutively active cyclin D1-CDK complexes, all in the absence of mitogen. Furthermore, these characteristics were abolished by the transient overexpression of the transducin heterotrimeric G protein α subunit strongly suggesting the transformation of G(o)α-ablated cells involves G(o)βγ subunits. This is the (first study to implicate a heterotrimeric G protein in tumor suppression.