In rhesus macaques, the concentration of immunoreactive prolactin in the amniotic fluid remains low during most of the first trimester of pregnancy and then increases abruptly at 60-80 days of gestation. During the second half of pregnancy, large amounts of prolactin accumulate in the amniotic fluid. Much of this amniotic fluid prolactin may originate from the superficial endometrium (decidua). This hypothesis is supported by the increasing amounts of decidual prolactin (dPRL) measured in endometrium obtained at early (50 days), mid-(80 days), and late (greater than or equal to 150 days) gestation. In culture, late pregnancy endometrium released more dPRL than did early pregnancy endometrium. When tissues were cultured in medium without progesterone, the amounts of dPRL measured in the medium declined steadily over 6 days, regardless of the gestational age of the endometrium. dPRL was consistently measured in medium harvested from cultures that received either progesterone or medroxyprogesterone; however, progesterone did not induce an increase in the amounts of dPRL released by cultures prepared from early pregnancy endometrium. This suggests that factors in addition to progesterone may stimulate the increase in dPRL that occurs at midgestation in rhesus macaques.