Granulosa cells of antral ovarian follicles from adult 5-day cyclic rats were cultured on each day of the cycle. The rat granulosa cell conditioned medium (rGCCM) was harvested and renewed on each day of a 4-day culture period. Inhibin-like activity and progesterone were estimated in rGCCM using an in-vitro bioassay system with dispersed rat anterior pituitary cells and radioimmunoassay respectively. Removal of steroids from rGCCM with dextran-coated charcoal was effective and did not significantly change the inhibin-like activity of the treated samples. On day 1 of culture the inhibin-like activity of rGCCM for each day of the oestrous cycle was 20-90% higher than on days 2, 3 and 4 of culture when low and constant levels were observed. Media collected after culture on days 1 and 2 from pro-oestrous cells contained larger amounts of inhibin-like activity than media collected on the other days of the cycle. On day 1 of culture, rGCCM from pro-oestrous cells contained higher concentrations of progesterone than that from cells collected on the other days of the cycle. On days 2, 3 and 4 progesterone levels in rGCCM were undetectable (less than 320 pmol/l) except in media from pro-oestrous cultures on day 2. Addition of FSH (62 micrograms/l) to granulosa cell cultures in medium with or without 10% fetal calf serum (FCS) did not alter the inhibin-like activity of rGCCM from pro-oestrous cells. The presence of FCS maintained the production of inhibin-like activity since rGCCM from cells cultured without FCS was devoid of FSH-suppressing activity after 3 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)