The capacity of human decidual tissue to synthesize prolactin de novo throughout late gestation was investigated and correlated with the levels of prolactin (PRL) in amniotic fluid. Maximal concentrations of PRL in both amniotic fluid and samples of decidua were found prior to the thirtieth week of gestation and declined simultaneously until term. A high correlation (r = 0.90, p < 0.00005) was found when the levels of PRL in amniotic fluid and the initial (preincubation) content of PRL in decidua from the same patient were compared. A very high correlation (r = 0.96, p < 0.00005) was seen between the ability of the decidua to produce prolactin in vitro and the corresponding levels of prolactin in amniotic fluid. No significant difference in any parameter tested was noted with respect to either the sex of the fetus or the mode of delivery (p > 0.05). These data are interpreted as indicating (1) that decidual tissue varies throughout late gestation, in both its initial content of prolactin and its ability to synthesize prolactin de novo, in a manner which correlates to a high degree with variations in amniotic fluid prolactin levels and (2) that the decidual tissue is the major source of amniotic fluid prolactin.