The effects of progesterone on decidual PRL (dPRL) production by human endometrium were investigated by culturing explants of proliferative (n = 20) and secretory (n = 12) endometrium in Dulbecco's Modified Eagle's Medium supplemented with 10 mM HEPES buffer, 0.1% gelatin (wt/vol), and antibiotics for 6 days and in medium containing 50 ng/ml of progesterone for 6-28 days. The culture medium was replaced daily, and the spent medium was assayed for dPRL. Representative explants were fixed for histological examination after 1, 2, 7, 14, and 28 days in vitro. When explants of endometrium obtained throughout the cycle were cultured in the presence of progesterone, dPRL production was stimulated. Proliferative endometria required 2-5 days in culture before dPRL was measured in the medium. Thereafter, dPRL production increased gradually for the duration of the experiment. Progesterone also induced glandular secretion and stromal differentiation (decidualization) in these tissues. Cultures of proliferative endometrium that did not receive progesterone did not produce detectable amounts of dPRL. When explants of secretory endometrium were cultured in DMEM without exogenous progesterone, dPRL was released into the medium for 2-5 days; however, dPRL production by the cultures that did not receive progesterone declined to undetectable levels, while that by the progesterone-treated cultures increased steadily. Explants of both proliferative and secretory endometrium that were fixed for histological examination after 28 days of culture in the presence of progesterone were composed predominately of large stromal cells that resembled the decidual cells of pregnancy endometrium.