Human decidua, chorion, amnion and placenta from the 1st., 2nd. and 3rd. trimesters of gestation were investigated for synthesis and secretion of prolactin by in vitro incubation of these tissue fragments in medium 199. Prolactin content in decidua was found to be significantly higher than that in chorion, amnion or placenta at any stages of gestation. During 6 hours of incubation, decidua secreted significantly more prolactin into medium than did chorion, amnion or placenta. The amount of prolactin secreted by decidua was significantly higher than the prolactin content in tissue before incubation. Decidua were also incubated in medium 199 with or without actinomycin-D (20-200 micrograms/ml), puromycin (200 micrograms/ml) or cycloheximide (100-200 micrograms/ml) for 12 hours. Both total prolactin secreted into medium and prolactin content in tissue after incubation were significantly lower than the control without inhibitor. Decidua of 2nd. trimester of gestation was noted to secrete more prolactin into medium than decidua of 1st. or 3rd. trimester of gestation. In further studies, 3H-leucine (200 muCi) was incubated for 12 hours with decidua (10 grams) from 2nd. trimester of gestation. The incorporated protein in medium or tissue was extracted, fractionated by gel filtration through Sephadex G-100 column and by 10% SDS-polyacrylamide gel electrophoresis. Peaks of 3H count and immunoreactive prolactin in gel slices were coincident with the peak of standard pituitary prolactin at gel slices' No. 31 (Rf = 0.62). These results demonstrate that prolactin is synthesized and secreted by human decidua, which is identical to human pituitary prolactin. The synthetic activity is most prominent in the decidua of the 2nd. trimester of pregnancy.