The time-dependent accumulation of prolactin (PRL) immunoreactivity in serum-free and in serum-supplemented cultures of bovine granulosa cells was observed. Blockade of endogenous progesterone by specific antiserum had no significant influence on PRL immunoreactivity, but progesterone addition (10-10,000 ng/ml) lead to a dose-dependent decrease. Antisera against testosterone and against estradiol-17 beta were inhibitors, and additions of these hormones (100-10,000 ng/ml) stimulated the release of PRL immunoreactivity. The results obtained suggest the secretion of a PRL-like peptide by bovine ovarian cells. Growth hormone, testosterone and estradiol may be stimulators, and progesterone a possible inhibitor of this process.