During the estrous cycle and early pregnancy, lymphohe-mopoietic cytokines and chemokines contribute to the regulation of ovarian function by orchestrating the recruitment and activation of leukocytes associated with the ovulatory follicle and corpus luteum. The purpose of this study was to investigate the physiological role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the ovary, utilizing mice genetically deficient in GM-CSF. Our results show that the mean duration of the estrous cycle in GM-CSF-deficient (GM−/−) mice was extended by 1.5 days (mean ± SE, 4.9 ± 0.3 vs. 6.5 ± 0.5 days for GM+/+ and GM−/− mice, respectively). Similar ovulation rates were observed in immature superovulated mice (31.8 ± 7.7 vs. 28.9 ± 6.4 oocytes per mouse) and adult naturally cycling mice (10.4 ± 0.8 vs. 10.3 ± 0.8 oocytes per mouse). Furthermore, comparable numbers of oocytes were released from GM+/+ and GM−/− ovaries in an in vitro perfusion model. However, ovaries in pregnant GM−/− mice were found to comprise fewer cells and synthesize less progesterone (141.6 ± 10.3 vs. 116.5 ± 6 nM plasma), although the duration of pseudopregnancy was unaltered by GM-CSF deficiency (11.0 ± 0.2 vs. 11.0 ± 0.5 days). Immunohistochemical staining of leukocytes in the ovary during the periovulatory period indicated that the size and composition of ovarian leukocyte populations were unaltered in the absence of GM-CSF. However, an effect of GM-CSF deficiency on the activation phenotype of ovarian leukocytes was indicated by a 57% increase in mean secretion of nitric oxide in in vitro-perfused GM−/− ovaries, and diminished major histocompability complex (MHC) class II (Ia) expression in ovarian macrophages and/or dendritic cells (30.5 ± 7.2% vs. 9.1 ± 1.8% positive stain in GM+/+ and GM−/− ovaries, respectively). Furthermore, ovarian macrophages and neutrophils were diminished in number after parturition, with significantly decreased CD11b+ (Mac-1) staining in the stromal region of postpartum GM−/− ovaries (6.7 ± 0.6 vs. 3.6 ± 0.7% positive stain). In summary, GM-CSF does not appear to be essential for ovarian function but may play a role in fine-tuning the activation status and adhesive properties of ovarian myeloid leukocytes. Aberrant activation of these cells appears to compromise the luteinization process and the steroidogenic capacity of the corpus luteum during early pregnancy in GM-CSF-deficient mice.