The aim of this study was to compare the pharmacodynamic response to a neuroactive steroid, pregnanolone, before and during different hormonal settings of postmenopausal hormone replacement therapy (HRT). Twenty-seven postmenopausal women with climacteric symptoms were administered HRT in a randomized, double-blinded, placebo-controlled crossover study. The women received 2 mg estradiol (E<sub>2</sub>) continuously during four 28-day cycles and 10 mg medroxyprogesterone acetate (MPA), 1 mg norethisterone acetate (NETA) or placebo sequentially for the last 14 days in each cycle. The pharmacodynamic response to pregnanolone was assessed before treatment and during the last week of each treatment, by comparing the effects of intravenous pregnanolone (3α-hydroxy-5β-pregnan-20-one) on saccadic eye velocity (SEV), saccade deceleration, saccade latency and self-rated sedation. Throughout the study daily symptom rating scales were kept. During the progesta gen phase of the treatment cycles, negative mood symptoms and physical symptoms were increased, whereas positive mood symptoms were decreased. Compared to pretretreatment conditions, E<sub>2</sub> alone did not change the responsiveness to pregnanolone. During progestagen addition to E<sub>2</sub>, the responsiveness to pregnanolone was increased. The sedation response increased compared to pretreatment conditions during both E<sub>2</sub> + MPA and E<sub>2</sub> + NETA treatment. Compared to E<sub>2</sub> treatment alone, addition of MPA increased the postpregnanolone effect on saccade deceleration, whereas the SEV response to pregnanolone was increased during E<sub>2</sub> + NETA treatment. It is concluded that pregnanolone sensitivity increases together with deterioration in mood symptoms during addition of progestagen to HRT.