An enriched rat Leydig cell preparation was preincubated with [C]arachidonic acid. Stimulation of the cells with arginine vasopressin (AVP) (1 µM) for 2 min caused a significant increase in labelled phosphatidic acid and a significant fall in radioactivity in phosphatidylinositol and phosphatidylinositol 4-monophosphate + phosphatidylinositol 4,5-bisphosphate. Preincubation with dibutyryl cyclic AMP had no effect on the AVP-induced phospholipid turnover. Leydig cells were preincubated with myo-[2-H]inositol for 22 h and then with 10 mM LiCl for 10 min. Exposure to AVP (1 µM) induced a rise in labelled inositol phosphates. The response was inhibited when the cells were preincubated with the phorbol ester, 4ß-phorbol 12-myristate 13-acetate (0.16 µM) for 10 min. These results provide evidence for an AVP-induced phospholipase C stimulation in rat Leydig cells and suggest a protein kinase C-dependent feedback inhibition of the stimulation. Other agonists that might have a regulatory function in the testis were tested for possible effects on phosphoinositide metabolism. Of prostaglandin E (10 µm), angiotensin II (0.1 µM), and bradykinin (0.9 µM), only the latter induced a significant increase in the labelled inositol phosphates. This suggests that Leydig cells possess a bradykinin receptor which can activate phospholipase C.