The aim of the present in-vitro study was to examine the role of obestatin in the direct control of basic avian ovarian granulosa cell functions proliferation, apoptosis and secretory activity. In addition, the effects of obestatin on hormone release by cultured ovarian granulosa cells and follicular fragments (containing both granulosa and theca cells) were examined. We identified the effect of obestatin addition (0.1, 10 or 100 ng/ml medium) on the accumulation of markers of proliferation (PCNA, cyclin B1, MAPK/ERK1,2) and nuclear (TdT) and cytoplasmic (bax, caspase 3) apoptosis, as well as the release of progesterone (P), testosterone (T) and estradiol (E) by cultured chicken granulosa cells. Furthermore, the action of obestatin addition (0.1, 10 or 100 ng/ml medium) on the release of P, T, E and argininevasotocin (AVT) by cultured fragments of chicken ovarian follicles was examined. The accumulation of proliferation and apoptosis markers was assessed by immunocytochemistry and SDS PAGE-Western immunoblotting. The release of hormones was determined by an EIA. It was observed that obestatin addition could inhibit the accumulation of proliferation markers (PCNA and cyclin B1, but not of MAPK/ERK1,2), promote the expression of nuclear (TdT) and cytoplasmic (bax, caspase 3) apoptosis markers and suppress P, T, and E release by cultured granulosa cells. In cultured ovarian follicular fragments, obestatin promoted P, T, and E, but not AVT, release. These observations represent the first demonstration that (i) obestatin can directly control avian ovarian cell proliferation, apoptosis and hormone release and (ii) the interrelationship between theca and granulosa cells can determine the characteristics of obestatin action on ovarian secretory activity.