Pretreatment of 1321N1 human astrocytoma cells with serum induces a pronounced increase in subsequent stimulation by forskolin and other agents of intracellular cyclic AMP accumulation, a phenomenon referred to as sensitization (Mol. Pharmacol. 39, 399-406, 1991). Pretreatment of these cells with lysophosphatidic acid induced sensitization to a similar extent as that with serum (approximately fivefold for forskolin stimulation and twofold for isoproterenol and prostaglandin E1 stimulation), with half-maximal effects at approximately 30 nM lysophosphatidic acid. Phosphatidic acid was effective but less potent whereas other lipids were ineffective. Sensitization by serum and by lysophosphatidic acid were almost completely inhibited by pertussis toxin pretreatment and partially inhibited by prolonged phorbol ester exposure to induce protein kinase C down-regulation. Among nine cell lines tested, those that exhibited sensitization with serum showed comparable sensitization with lysophosphatidic acid. The effects of both lysophosphatidic acid and serum were markedly inhibited by treatment with phospholipase B but only minimally altered with phospholipases A2, D, and C. Exposure of cells to phospholipase C alone induced approximately threefold sensitization, but both serum and lysophosphatidic acid were able to induce further three- to fourfold sensitization above that induced by phospholipase C alone. In contrast, the effects of serum and lysophosphatidic acid were not additive with each other. Together these results suggest that lysophosphatidic acid or a closely related compound present in serum is the factor responsible for sensitization of the cyclic AMP pathway.