To determine whether human decidua and/or chorion synthesizes and secretes prolactin, explants of decidua obtained at Caesarian section and explants of chorion from the membranes separating dizygotic twins were cultured for periods of up to 6 days. The decidual explants released 366 +/- 37 ng prolactin/100 mg tissue (mean +/- S.D.) during each day in culture and incorporated 3H-labelled amino acids into immunoprecipitable prolactin. In the radioimmunoassay for prolactin, serial dilutions of incubation medium displaced 125I-labelled prolactin parallel to the displacement by pituitary prolactin and the prolactin in the medium eluted from Sephadex G-150 in a position indentical to that of pituitary prolactin. Chorionic explants released prolactin into the incubation medium during day 1 of culture only and did not incorporate 3H-labelled amino acids into prolactin. These results demonstrate that prolactin is synthesized by the decidua and not by the chorion and suggest that the decidua is the source of prolactin in amniotic fluid.