Cultured pituitary cells from adult female rats were treated for 4 or 24 h in the absence or presence of E2 (10−9 mol/1) with increasing concentrations of the transcription inhibitor actinomycin-D or the translation inhibitor puromycin. During the last 4 h of incubation, LH release was stimulated with 5 × 10−10 mol/1 GnRH. The positive E2 effect observed after 24 h treatment with the steroid was clearly abolished by actinomycin-D at concentrations ≥10−10 mol/1 and by puromycin at concentrations ≥ 10−5 mol/1. These findings indicate that the positive E2 effect on GnRH-induced LH release is fully dependent on intact mRNA and protein synthesis. The negative E2 effect observed after 4 h treatment with the steroid was not affected by actinomycin-D and abolished by puromycin only at concentrations ≥ 10−4 mol/1. Similar results were obtained, when cells had been treated for 4 h with actinomycin-D or puromycin before the 4 h E2 treatment started. Thus, the negative E2 effect seems to be independent of mRNA synthesis and dependent on protein synthesis to a lesser extent than the positive E2 effect. In an attempt to identify positively the subcellular mechanism via which E2 exerts its negative effect, several steps in the GnRH stimulus secretion coupling mechanism were checked whether or not they are modulated by E2. The negative effects of E2 (10−9 mol/1) on LH release induced by GnRH (10−10, 10−9 mol/1) and by the activators of voltage dependent Ca2+ channels K+ (64 mmol/1) or veratridine (3.3 × 10−5 mol/1) were comparable to those of the calcium antagonist verapamil (10−6 mol/1). These findings supported the speculation that E2 might act on the Ca2+ channels. The LH release induced by the Ca2+ ionophores A 23 187 (10 −4 mol/1) or ionomycin (6.6 × 10−5 mol/1), however, was also significantly reduced by 10−9 mol/1 E2, indicating that the steroid modulated a mechanism secondary to the increase of intracellular Ca2+. Also GnRH (10−9, 10−8 mol/1) induced accumulation of [3H]inositol phosphates was not influenced by E2 (10−9 mol/1) treatment, though the steroid exerted a significant negative effect on the LH release by these cells, indicating that phosphatidylinositol-4,5-biphosphate breakdown is not the point of attack for the estrogen. The LH release induced by the activation of protein kinase C with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 10−7 mol/1) or with the synthetic diacylglycerol 1,2-dioleyl-sn-glycerol (DOG, 5 × 10−5 mol/1, 10−4 mol/1) was significantly reduced by E2 treatment. These findings give rise to the speculation that E2 might exert its negative effect on GnRH-induced LH release in cultured pituitary cells at the level of protein kinase C or a mechanism secondary to the activation of this enzyme.