Correlations of the rise in prostaglandin F2 alpha (PGF2 alpha) levels in human endometrium during the menstrual cycle with changes in plasma concentrations of ovarian steroids have suggested that progesterone (P) priming of the endometrium is necessary for the stimulation of PGF2 alpha production by estradiol (E2). However, despite the absence of significant levels of P in plasma during the follicular phase of the cycle, PGF2 alpha output by epithelial cell cultures derived from proliferative endometrium was stimulated in vitro by 10(-8) M E2 at least as well as PGF2 alpha output from secretory endometrium. In addition, basal PGF2 alpha output by proliferative endometrium under organ culture conditions was significantly greater than that by secretory endometrium. Concurrent addition of P counteracted the effects of E2 in these in vitro systems. P (10(-7) M) plus E2 (10(-8) M) resulted in PGF2 alpha output as low or lower than those of control incubations of secretory and proliferative endometrium. In the epithelial cell cultures, significant net stimulation by 10(-8) M E2 occurred in the presence of 10(-6) M P, a noteworthy finding since the rise in endometrial PGF2 alpha during the luteal phase takes place when the tissue is exposed to both E2 and P. P lowered the basal output of PGF2 alpha by endometrium in organ culture, but not by epithelial cells in monolayer cultures. E2 appears to stimulate PGF2 alpha output by increasing synthesis rather than diminishing metabolism, since exogenous [3H]PGF2 alpha was metabolized to an equivalent extent by fragments of secretory endometrium or glandular epithelial cells regardless of whether E2 was added to the incubation medium. The results from this study confirm that only secretory endometrium responds to E2 in vitro by significantly increasing PGF2 alpha output. The lack of response by proliferative endometrium, when contrasted with the marked responsiveness of epithelial cells derived from this tissue, suggests that an inhibitory influence is removed during the isolation or subsequent culture of endometrial glands.