Arginine-vasopressin (AVP) stimulated the formation of labelled phosphatidic acid (PA) in [C]arachidonic acid-prelabelled rat Leydig cells. After addition of 10 M AVP [C]arachidonoylphosphatidic acid reached a maximum within 2 min. The increase was dose-dependent (10-10 M). No change in labelling of other phospholipids and diacylglycerol could be detected. The V antagonist dPTyr(Me)AVP inhibited in a dose-dependent manner the AVP-stimulated accumulation of PA. The V agonist dPVDAVP was without effect. The present results suggest that AVP binds to V receptors in rat Leydig cells resulting in stimulation of PA turnover. We suggest that the AVP-stimulated PA formation is an indication of phosphoinositide turnover.