The Gi class of heterotrimeric G proteins has been implicated in transmitting mitogenic signals from a variety of seven-transmembrane domain receptors. In addition, the alpha subunit of Gi2 (alpha i2) is oncogenic when mutated to a constitutively active form (gip2). The mechanism by which Gi2 stimulates cellular proliferation is unknown, but is believed to involve activation of the mitogen-activated protein kinase (MAPK) signaling cascade. To study Gi2 activation of the cascade, we transiently expressed a mutant, pertussis toxin (PTX)-resistant alpha i2 in Chinese hamster ovary cells. After PTX treatment of these cells, Gi-coupled receptors specifically activated PTX-resistant Gi2 without activating other Gi proteins. Receptor-mediated activation of Gi2 led to activation of MAPK and its upstream activator, MAPK/ERK-activating kinase (MEK). Activation of MAPK and MEK by Gi2 was blocked by expression of a dominant-negative mutant of Ras. Gi2 activation did not, however, detectably increase the proportion of Ras protein in the GTP-bound form. Additional experiments suggest that Gi2 stimulates the MAPK pathway, at least in part, by mechanisms that involve release of its beta gamma subunit, as well as activation of phosphatidylinositol-3 kinase.