To explore the direct effect of estradiol-17 beta on pig luteal cells and its ability to counteract a gonadotropic stimulus, luteal cells were incubated in vitro in the presence of estradiol alone, human chorionic gonadotrophin (hCG) alone or estradiol combined with hCG. Luteal cells were collected at three different stages of the luteal phase (0-3 days after ovulation, early; 8-10 days after ovulation, mid; and 14-16 days after ovulation, late). Estradiol alone did not exert any effect on progesterone production by any of the 3 types of luteal cells. Progesterone production by cells in the mid-luteal phase was enhanced by hCG in a dose dependent manner. High doses of estradiol combined with hCG significantly diminished production of progesterone by cells collected during the early luteal phase. The strongest inhibitory effect of estradiol on hCG stimulated progesterone production was observed in cultures of luteal cells in the midluteal phase. In contrast, 100 ng of estradiol and the 10 and 100 ng/ml concentrations of hCG increased progesterone secretion over basal control values in cultured luteal cells in the late luteal phase. Differences in the production of progesterone in response to estradiol at different stage of the luteal phase suggest that different cellular mechanisms must be triggered in each luteal phase investigated. These data support a physiological role for estrogen in the regulation of the pig corpus luteum during its life span in the non-fertile cycle.