Many receptors for neuropeptides and hormones are coupled with the heterotrimeric G_i protein, which activates the p42/44 mitogen-activated protein kinase (ERK/MAPK) cascade through both the α- and βγ-subunits of G_i (–). The βγ-subunit activates the ERK/MAPK cascade through tyrosine kinase^,,. Constitutively active Gα_i2 (gip2) isolated from adrenal and ovarian tumours, transforms Rat-1 fibroblasts and also activates the ERK/MAPK cascade by an unknown mechanism,. The ERK/MAPK pathway is activated by Ras, and is inhibited when the low-molecular-mass GTP-binding protein Rap1 antagonizes Ras function. Here we show that a novel isoform of Rap1 GTPase-activating protein, called rap1GAPII, binds specifically to the α-subunits of the G_i family of heterotrimeric G-proteins. Stimulation of the G_i-coupled m2-muscarinic receptor translocates rap1GAPII from the cytosol to the membrane and decreases the amount of GTP-bound Rap1. This decrease in GTP-bound Rap1 activates ERK/MAPK. Thus, the α-subunit of G_i activates the Ras-ERK/MAPK mitogenic pathway by membrane recruitment of rap1GAPII and reduction of GTP-bound Rap1.