Lipo-chitooligosaccharides (Nod factors) are produced by symbiotic Rhizobium sp. bacteria to elicit Nod responses on their legume hosts. One of the earliest responses is the formation of phosphatidic acid (PA), a novel second messenger in plant cells. Remarkably, pathogens have also been reported to trigger the formation of PA in nonlegume plants. To investigate how host plants can distinguish between symbionts and pathogens, the effects of Nod factor and elicitors (chitotetraose and xylanase) on the formation of PA were investigated in suspension-cultured alfalfa (Medicago sativa) cells. Theoretically, PA can be synthesized via two signaling pathways, i.e. via phospholipase D (PLD) and via phospholipase C in combination with diacylglycerol (DAG) kinase. Therefore, a strategy involving differential radiolabeling with [(32)P]orthophosphate was used to determine the contribution of each pathway to PA formation. In support, PLD activity was specifically measured by using the ability of the enzyme to transfer the phosphatidyl group of its substrate to a primary alcohol. In practice, Nod factor, chitotetraose, and xylanase induced the formation of PA and its phosphorylated product DAG pyrophosphate within 2 min of treatment. However, whereas phospholipase C and DAG kinase were activated during treatment with all three different compounds, PLD was only activated by Nod factor. No evidence was obtained for the activation of phospholipase A(2).