Cells with the ultrastructural features of macrophages were seen within corpora lutes of mice shortly after induction of ovulation with gonadotropins. Upon isolation, mononuclear phagocytes from luteal tissue had histochemical and functional properties characteristic of macrophages from other body sites. These attributes included intense staining for nonspecific esterase activity, phagocytosis of IgG-coated red blood cells via an Fc receptor, ingestion of latex beads, and trypsin-resistant adherence to the surface of culture dishes. Furthermore, the ovarian macrophages specifically bound to their surface a monoclonal antibody against a plasma membrane protein of mononuclear phagocytes. When mouse granulosa cells were co-cultured with peritoneal macrophages, progesterone secretion was increased, and the magnitude of the increased steroidogenesis was directly related to the number of mononuclear phagocytes added to the cultures. Likewise, cultured luteal cells from superovulated mice secreted more progestins in the presence of peritoneal macrophages. Luteal cells also produced more progesterone when they were recombined in culture with autologous macrophages from corpora lutea. The mononuclear phagocytes from ovarian tissue appeared to have a greater ability to stimulate luteal cells than did peritoneal macrophages. Conditioned media from cultured macrophages failed to stimulate steroid secretion by granulosa cells, suggesting that close proximity of the two cells is required for the effect on luteal cell function. Thus, it appears that macrophages within corpora lutea may play a role in maintaining progesterone secretion by luteal cells.