Ovarian functions are regulated by a wide variety of substances of hypothalamic, pituitary and intraovarian origin. In particular, prolactin (PRL) plays an important role in the control of ovaries. The aim of our in-vitro experiments was to prove a possibility of PRL production by bovine granulosa cells and to search into the endocrine regulators of this process. In the course of experiment 1 it was observed that the marked time-dependent accumulation of immunoreactive PRL took place during long-time cultivation of granulosa cells both in serum-free and in serum-dependent medium. After 12-24 hours of cultivation this level was reduced, but after 120 hours of cell culture the medium PRL-immunoreactivity gradually rose to exceed the starting level 2.1-2.4 times. FSH additions (10-10,000 ng/ml) led to a dramatical rise of PRL-immunoreactivity in a dose-dependent manner. A greater increase in FSH doses (1000 or 10,000 ng/ml) activated this process 14.0-18.0 times. In the other experiments the effects of LH, LH-RH and various nonapeptide hormones on the PRL-like substance production were investigated. LH stimulated PRL-like substance production at a great dose only (10 IU/ml). The lower doses did not have any significant influence on the process. Low doses of oxytocin (1 or 10 IU/ml) blocked, and higher doses (100-1000 IU/ml) stimulated the granulosa PRL-like production. Arginine-8-vasopressin (AVP) (1-1000 ng/ml), arginine-8-vasotocin (AVT) (10-1000 ng/ml), or LH-RH (10-10,000 ng/ml) failed to influence the immunoreative PRL accumulation in the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)