To examine the production of cytokines by the ovary during ovulation, ovaries were obtained from immature rats and from eCG/hCG-primed immature rats at different stages of the ovulatory process (before hCG injection, 10 h after hCG, and 20 h after hCG) and were perfused in vitro for 5 h. Large quantities of interleukin (IL)-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) bioactivity and smaller amounts of tumor necrosis factor-alpha (TNF alpha) and IL-1 bioactivity were found in the perfusate. IL-2 and IL-3 were not detectable in the perfusion media. The GM-CSF content was significantly higher in the perfusate of ovulating ovaries (obtained 10 h after hCG) compared to the earlier stages. Studies on preovulatory ovaries (prior to hCG injection) revealed that GM-CSF release was not influenced by LH, but was markedly increased when recombinant human IL-1 beta (4 ng/ml) was added to the perfusion medium. IL-6 was released in similar amounts from ovaries at all stages. The identity of bioactive GM-CSF was confirmed by neutralization with a specific polyclonal antibody against murine GM-CSF. Size-exclusion chromatography of perfusion medium revealed peaks of GM-CSF and IL-6 bioactivity at approximate molecular masses of 21-23 kDa and 24-25 kDa, respectively. This study demonstrates that the rat ovary produces IL-6, GM-CSF, TNF alpha, and IL-1 prior to and during the ovulatory process and that there are temporal fluctuations in GM-CSF release with a peak in output at ovulation.