OBJECTIVE The purpose of this study was to determine whether interferon gamma affects luteinized human granulosa cell progesterone, estrone, and estradiol production in the presence and absence of associated white blood cells by either cytotoxic or antiproliferative mechanisms. METHODS Luteinized granulosa cells were isolated by Percoll centrifugation from women during in vitro fertilization cycles. Some cell suspensions were further treated with anti-CD45 magnetic immunobeads to remove associated white blood cells. Granulosa cells with and without white blood cells were cultured in the presence of interferon gamma (0.5 to 50 ng/ml) for 48 hours. Medium was changed at 24-hour intervals, and spent medium was assayed for progesterone, estrone, and estradiol. In separate experiments granulosa cell viability was assessed with the tetrazolium salt reduction assay. RESULTS Interferon gamma significantly inhibited granulosa cell progesterone production in both basal and human chorionic gonadotropin-stimulated cells cocultured with white blood cells in a concentration-dependent manner, whereas cells cultured free of white blood cells demonstrated less inhibition. In the absence of interferon gamma a more profound increase in granulosa cell progesterone synthesis was found in human chorionic gonadotropin-stimulated cultures without associated white blood cells. Interferon gamma inhibited granulosa cell estrone and estradiol production in basal cultures containing white blood cells in both a time- and concentration-dependent manner. Estrone production was not affected by interferon gamma in human chorionic gonadotropin-stimulated granulosa cell cultures containing white blood cells, whereas estradiol secretion was decreased at 48 hours with 50 ng/ml interferon gamma. Both estrone and estradiol synthesis were inhibited by 50 ng/ml interferon gamma in granulosa cell cultures free of white blood cells. In cultures free of interferon gamma, granulosa cell estrone and estradiol secretion was not affected by human chorionic gonadotropin stimulation compared with basal controls regardless of the presence or absence of white blood cells. All concentrations of interferon gamma used had no effect on granulosa cell viability at any time point tested. CONCLUSIONS Our data suggest that interferon gamma affects granulosa cell steroid production both independently and in synergy with associated white blood cells and further supports the hypothesis that interferon gamma may be an important intraovarian regulator of ovarian steroid production during the luteal phase.