Epidermal growth factor (EGF) stimulates progesterone production by human granulosa--luteal cells in culture. The present study investigated some of the parameters that affect the magnitude of human granulosa--luteal cells' response to EGF. Cells from pre-ovulatory follicles obtained 36 h post-human chorionic gonadotrophin (HCG) were cultured for 12 days with or without EGF (20 ng/ml). Medium was changed every 48 h and assayed for progesterone by radioimmunoassay. DNA content of the cultured cells was determined fluorometrically. EGF was added every other day to the culture medium, starting on either day 4, 6 or 8 of culture, up to day 10, and compared with controls. When EGF was initiated on day 4, the medium had significantly higher progesterone concentration than control samples on days 6, 8, 10 and 12 of culture (P < 0.01). When EGF was withheld until day 6 or 8, progesterone concentrations were not significantly higher than control values. When EGF was added on day 4 and discontinued on day 8 or 10, progesterone concentrations were reduced significantly (P < 0.001) compared with the group where EGF was added continuously from day 4 to 10. These data suggest that: (i) human granulosa--luteal cells require the early exposure and continuous presence of EGF for the stimulatory effect on progesterone secretion, (ii) cells not exposed initially to EGF do not respond in a similar way, (iii) EGF is capable of maintaining progesterone production for a period > 12 days. Therefore, normal luteal function may require the early and continuous presence of EGF.