Mutationally activated alpha subunits of two G proteins, Gs and Gi2, induce neoplastic transformation of fibroblasts and are found in human tumors. Here we report that mutationally activated alpha subunits of two other G proteins, G12 and G13, induce neoplastic transformation of Rat-1 fibroblasts and NIH3T3 fibroblasts. Constitute activation of these alpha subunits resulted from replacement by leucine of glutamine-229 and glutamine-226 in alpha 12 and alpha 13, respectively. Transient expression of mutant alpha 12 and alpha 13 cDNAs induced focus formation in Rat-1 cells and NIH3T3 cells, and stable expression of these mutant proteins in Rat-1 cells accelerated growth rate, induced growth in soft agar, and increased DNA synthesis. Mitogen-activated protein (MAP) kinase activity, stimulated by EGF, was increased in Rat-1 cells that expressed mutant alpha 12 or alpha 13. The MAP kinase cascade plays a role in mediating neoplastic transformation induced by other GTPases, including ras and the alpha subunit of Gi2. Therefore, we propose that the MAP kinase cascade is an effector pathway affected by alpha 12 and alpha 13 and may contribute to neoplastic transformation by these mutant proteins. We predict that activating somatic mutations in alpha 12 and alpha 13 genes will be found in human tumors, as is the case for mutationally activated alpha subunits of Gs and Gi2.