The ovulatory process has been compared with inflammation because several classical inflammatory mediators appear to participate in this process. One component of the inflammatory reaction is the migration of leukocytes to the site of inflammation and the subsequent activation of these cells. We have reported recently that perfusion of leukocytes into the rat ovary in vitro enhances the number of LH-induced ovulations, which suggests an active role of leukocytes in ovulation. In the present study we characterize immunohistochemically the distribution of macrophages, T lymphocytes, and granulocytes in the ovaries of untreated immature rats and of eCG-hCG-primed rats killed prior to hCG injection, at ovulation, and at 33-36 h post-ovulation. Macrophages, identified with monoclonal antibodies ED1 and ED2, were the major leukocyte population and were found primarily in the medullary region surrounding the blood vessels. The density of the cells in this region increased continuously during development to sexual maturity and until after ovulation. Macrophages were also present in the thecal layer of the preovulatory follicles, and the numbers of these cells increased about 5-fold in this area in ovulating follicles (12 h after hCG) compared to preovulatory follicles (before hCG). A portion of macrophages in both areas expressed major histocompatibility complex (MHC) class II antigens (OX6+); these cells were present mostly in the medullary region, with no apparent change in density during the periovulatory period. Neutrophilic granulocytes comprised a lesser proportion of the total leukocyte population in the medullary region but were abundant in the thecal layer. The density of neutrophils increased 3-fold in the medullary region and 8-fold in the thecal region in ovulatory compared to preovulatory follicles. T lymphocytes (OX52+) were evenly distributed at relatively low density in the medulla and the stroma of the cortex. Most T lymphocytes expressed the CD8 antigen (OX8+) and hence were of the MHC class I-restricted phenotype. Few T lymphocytes were present in the thecal layer. In summary, macrophages, neutrophilic granulocytes, and T lymphocytes are present in the ovary at ovulation. There is a selective increase in the numbers of macrophages and neutrophilic granulocytes in the medullary region and in the thecal layer as the ovulatory period progresses, indicating that these cells may actively be involved in the tissue remodeling occurring at ovulation.