The effect of human chorionic gonadotropin (hCG) on intact Leydig cell phospholipid methylation was studied. Hormonal stimulation of rat Leydig cells increased the incorporation of [methyl-3H]methionine into phospholipids threefold. This effect was observed after 10 minutes of incubation time and was time and dose dependent with a maximal stimulation at 67 ng/ml of hCG. In the presence of hCG, 3H-labeled methyl groups were preferentially incorporated into phosphatidyl-N-monomethylethanolamine. This effect of hCG was not reproduced by dibutyryl cyclic adenosine monophosphate (cAMP), cholera toxin, or forskolin. Purified hCG beta subunit but not hCG alpha subunit had stimulatory activity on Leydig cell phospholipid methylation. We conclude that luteinizing hormone (LH)/hCG stimulates specifically Leydig cell phospholipid methylation, because LH-releasing hormone or [Arg8]-vasopressin did not modify these reactions. We postulate that these reactions are occurring at a cellular level that involves hormone-receptor interaction. It is also suggested that this biological response involves hCG beta subunit receptor interaction and does not require cAMP synthesis.