Several studies have shown the potential role of phosphatidic acid (PA) as a second messenger in different cell types. Thus, PA has been shown to mimic physiological agonists leading to various cellular responses, such as neurotransmitter and hormone release, cell proliferation by modulating DNA or RNA synthesis, the expression of several proto-oncogenes and growth factors, and the stimulation of enzyme activities such as phospholipase C (PLC), protein kinases and cyclic AMP (cAMP) phosphodiesterase. Stimulation of [3H]arachidonate-labelled rat thymocytes with the mitogen lectin concanavalin A (con A) resulted in enhanced production of radiolabelled PA after only 5 min of activation. The radiolabelled PA increase corresponded to a real increase in PA mass as determined by GLC quantification of its fatty acid content. In the presence of ethanol (0.5%), formation of phosphatidylethanol was not observed after 5 min of con A activation. Pretreatment of cells with R 59022 (10 microM), a diacylglycerol (DAG) kinase inhibitor, showed an inhibition in the formation of radiolabelled PA and in PA mass. These results suggest that the PLC-DAG kinase may be the pathway for PA synthesis in the first minutes of mitogenic thymocyte activation. A detailed analysis of the fatty acid composition showed that the relative amount of unsaturated fatty acids was increased in PA from stimulated cells concomitantly with a decrease in saturated ones; in particular, arachidonic acid was increased approximately 2-fold only 2 min after con A addition whereas palmitic acid was decreased for the whole period investigated (20 min). These changes favour the hydolysis of phosphoinositides rather than phosphatidylcholines by PLC. As PA remains a minor phospholipid, these changes are unlikely to affect cell membrane fluidity; but PA being now well recognized as a potential second messenger, its increased content as well as its increased unsaturation in the fatty acyl moiety might modulate several signalling pathways or the activity of enzymes such as cyclic nucleotide phosphodiesterase, controlling in this way the cellular level of cAMP, a negative regulator of blastic transformation.