3-Deazaadenosine (3-DZA), an inhibitor of somatic cell transmethylations, inhibited in vitro HCG-stimulated testosterone synthesis by rat testis interstitial cells. A maximal inhibition of 50% was observed with 100 microM 3-DZA; in addition homocysteine-thiolactone (Hcy) enhanced the inhibitory effect of 3-DZA. On the other hand, when cells were stimulated with dibutyryl cyclic AMP (Bt)2-cAMP, 3-DZA did not exert any effect on the stimulation. The presence of 3-DZA in the incubation medium neither modified HCG Kd values nor the number of its binding sites to Leydig cells. These results demonstrate that inhibitors of transmethylation reactions interfere with hormone-stimulated testosterone synthesis, suggesting that those reactions (presumably phospholipid methylation) at the plasma membrane level are involved in hormone-stimulated testosterone synthesis by rat Leydig cells.