In nonbroody birds, participation of prolactin in the reproductive functions is still unknown and its role in the local regulation of ovarian activity has had little attention. Therefore, the aim of the present study was to determine whether in the domestic hen prolactin influences in vitro steroid secretion by white and yellow chicken ovarian follicles. Small white (1-4 mm), medium white (4-6 mm), large white (6-8 mm) and 3 largest yellow preovulatory follicles (F3-F1; F3<F2<F; 25-36 mm) were isolated at stage 22 h and 3 h before ovulation of the largest (F1) follicle. From the preovulatory follicles, granulosa and theca layers were separated and divided into 4 pieces. Whole white follicles (6 small/dose/ovary; 1 medium or 1 large/dose/ovary) or parts of the granulosa or theca layers were randomly assigned to 1 ml of Eagle's medium containing 0 (control), 1, 10 or 100 ng/ml ovine prolactin and were incubated for 24 h at 38 degrees C. Following incubation, the medium was collected for estradiol and progesterone determination (RIA), and tissues of the follicular wall for protein assay by the method of Lowry. It was found that prolactin affects steroid secretion by chicken ovarian follicles. In white follicles prolactin inhibits estradiol secretion, whereas in yellow preovulatory follicles it stimulates or inhibits steroid secretion and its activity depends on: (1) the dose ofprolactin, (2) the type of the follicular layer secreting steroids, (3) the position of the follicle in the hierarchy and (4) the stage of the ovulatory cycle.