Follicular fluid samples were obtained from the largest pre-ovulatory follicle of 120 women undergoing in-vitro fertilization and were examined for melatonin by enzyme-linked immunosorbent assay and the steroids oestradiol and progesterone by radioimmunoassay. The concentrations (mean +/- SE) of melatonin (213.4 +/- 18.9 pmol/l) and progesterone (20.1 +/- 1.1 mumol/l) in follicular fluid during the autumn and winter (dark) months were significantly higher than during the spring and summer (light) months, melatonin (138.4 +/- 12.5 pmol/l) and progesterone (11.6 +/- 0.8 mumol/l). By contrast, oestradiol concentrations were significantly lower during the dark months than during the light months (264.7 +/- 44.1 and 661.8 +/- 55.1 nmol/l respectively). There was a positive correlation between follicular fluid melatonin and progesterone concentrations (r = 0.271, P < 0.05, n = 120) and a negative relationship between melatonin and oestradiol (r = -0.254, P < 0.05, n = 120). The effects of melatonin alone and in combination with human chorionic gonadotrophin (HCG) or follicle stimulating hormone (FSH) on steroidogenesis by human granulosa cell culture were also investigated. Melatonin had minimal effects on oestradiol or progesterone production by granulosa cells. Interestingly, the oestradiol response in culture appeared to be different according to the time of the year when harvested. During the light period oestradiol production was enhanced. Melatonin also synergized with HCG in increasing progesterone production on days 6 and 7 after treatment during both light and dark periods. FSH stimulated oestradiol production by the cells on day 2 of culture. Melatonin had no effect on FSH stimulation of oestradiol production. The results of this study suggest that melatonin may be involved in the regulation of steroidogenesis by the human ovaries.