Expression of the GTPase-deficient, activated mutant alpha-subunit of the heterotrimeric G protein G12 (Galpha12QL) leads to the neoplastic transformation of fibroblast cell lines. The mitogenic pathway regulated by Galpha12QL includes an extensive signaling network involving several small GTPases and various kinases. In addition, Galpha12QL has been shown to potentiate the serum-induced phospholipase-A2 activity in NIH3T3 cells. In the present study, we demonstrate that cycloxygenase-2 (COX-2) pathway is involved in the mitogenic pathway activated by Galpha12QL. Expression of Galpha12QL and not Galpha13QL, stimulates the serum-induced release of arachidonic acid in NIH3T3 cells. Furthermore, expression of Galpha12QL or the stimulation of wild-type Galpha12 induces the expression of COX-2. Our results also indicate that the COX-2 inhibitor acutely disrupts the DNA-synthesis stimulated by Galpha12QL in NIH3T3 cells. These studies, for the first time, identify the crucial role of COX-2 in Galpha12-mediated regulation of cell proliferation and suggest a role for prostaglandin-derived autocrine loop in Galpha12-mediated signaling pathways.