Lysophosphatidate (LPA), the simplest natural phospholipid, is highly mitogenic for quiescent fibroblasts. LPA-induced cell proliferation is not dependent on other mltogens and is blocked by pertussis toxin. LPA initiates at least three separate signaling cascades: activation of a pertussis toxin-insensitive G protein mediating phospholnosltlde hydrolysis with subsequent Ca2+ mobilization and stimulation of protein kinase C; release of arachidonic acid in a GTPdependent manner, but independent of prior phosphoinositide hydrolysis; and activation of a pertussis toxinsensitive 0, protein mediating inhibition of adenylate cyclase. The peptide bradykinin mimics LPA in inducing the first two responses but fails to activate GI and to stimulate DNA synthesis. Our data suggest that the mltogenlc action of LPA occurs through GI or an?-lated pertussis toxin substrate and that the phosphoinositide hydrolysis-protein kinase C pathway is neither required nor sufficient, by itself, for mitogenesis. The results further suggest that LPA or LPA-like phospholipids may have a novel role In G protein-mediated signal transduction.