Phospholipase D, an enzyme involved in signal transduction cascades, catalyses the formation of phosphatidic acid and, when ethanol is present, the formation of phosphatidylethanol. In the present study we demonstrate that stimulation of muscarinic acetylcholine receptors as well as P2-purinergic receptors induces activation of phospholipase D in primary cultures of astroglial cells. Both the hydrolysis and the transphosphatidylation reactions were stimulated by receptor agonists. Carbachol and ATP induced a rapid increase in the amount of [3H]phosphatidic acid in astroglial cells prelabelled with [3H]oleic acid. When ethanol (150 mM) was present, phosphatidylethanol was formed. Furthermore, the receptor-mediated increase in the concentration of phosphatidic acid was inhibited by ethanol, indicating that the phosphatidic acid production was indeed mediated by phospholipase D. The formation of phosphatidylethanol was concentration dependent, with a half-maximal effective concentration of 5 x 10(-5) M for carbachol and 10(-5) M for ATP. The carbachol-induced response was almost completely inhibited by atropine, indicating activation of phospholipase D via muscarinic receptors. The purinergic response is most probably mediated via P2-receptors since ADP was almost as efficient as ATP in inducing phosphatidylethanol formation, whereas AMP was significantly less potent. We conclude that astroglial cells in primary culture display muscarinic and purinergic receptors coupled to phospholipase D. The relationship to cell function needs to be further investigated.