Transforming growth factor- (TGF-) is a potent inducer of numerous extracellular matrix components, largely through a transcriptional mechanism. To define the postreceptor signaling pathways used by TGF- in the induction of extracellular matrix gene expression, we have utilized the human lung carcinoma cell line, A549, in transfection experiments with the TGF- inducible reporter construct, p3TP-Lux. Previous work from this laboratory using pharmacologic agents suggested that a phosphatidylcholine-specific phospholipase C and protein kinase C may be involved in early aspects of TGF- signaling. Here we provide evidence that TGF- induces a rapid and transient increase in diacylglycerol (DAG) production. When cells transfected with the p3TP-Lux reporter plasmid are simultaneously treated with TGF- and a DAG kinase inhibitor, we observed a higher level of luciferase than with TGF- alone. We also find elevated levels of phosphocholine in cells following TGF- treatment. Further, exogenously added bacterial phosphatidylcholine phospholipase C (PC-PLC) is capable of inducing expression of the p3TP-Lux reporter to the same extent as TGF- indicating that the bacterial PC-PLC can mimic the TGF- effect. In contrast, neither hexanoyl sphingosine (a ceramide analogue) nor arachadonic acid induce expression of the p3TP-Lux reporter. Measurements with the fluorescent, calcium-sensitive dye, FURA2, indicated that there was no change in intracellular calcium in response to TGF-. Furthermore, buffering intracellular calcium with the calcium chelating agent BAPTA/AM failed to block TGF- induction of the p3TP-Lux reporter. Thus the TGF- signaling pathway appears to involve the production of diacylglycerol but is independent of calcium.