Allopregnanolone is a neuroactive steroid involved in modulating behavioral functions, stress, and neuroendocrine axes in rats. Changes in plasma allopregnanolone levels throughout the menstrual cycle have been reported in healthy women, but there exists no information on the possible gender or age-related changes or on the source(s) of circulating allopregnanolone. The aim of the present study was to assess serum allopregnanolone concentrations according to gender, menstrual cycle, age, and menopause in normal men and women; serum progesterone (P) and dehydroepiandrosterone (DHEA) levels were evaluated in the same specimens. In addition, the possible source of circulating allopregnanolone in fertile women was investigated by using stimulatory and inhibitory endocrine tests acting on the ovary and/or adrenal cortex. The present study included 189 fertile women, 112 postmenopausal women, and 46 men. Serum steroid levels were determined after extraction, using specific RIAs. Allopregnanolone levels in fertile women in the follicular phase were similar to those in age-matched men; no significant difference was found between fertile women in the follicular phase and postmenopausal women. The highest levels were found in fertile women during the luteal phase (P < 0.01). An age-related decrease was observed in men (P < 0.01), but not in women. P and DHEA levels were significantly higher in women than in men and were higher in fertile women than in postmenopausal women (P < 0.01). Both P and DHEA showed an age-related decrease in men and women (P < 0.01). Serum allopregnanolone and P, but not DHEA, significantly increased in response to a GnRH test, whereas corticotropin-releasing factor and ACTH tests elicited a significant increase in allopregnanolone, P, and DHEA levels (P < 0.01). The suppression of adrenal steroidogenesis by dexamethasone markedly reduced both allopregnanolone and DHEA serum levels (P < 0.01). In conclusion, the present study demonstrated that although men show an age-related decrease, serum allopregnanolone levels in women do not change with age and correlate with P levels during the menstrual cycle and in response to endocrine tests. Ovary and adrenal cortex may be major sources of circulating allopregnanolone in fertile women.