The release of human chorionic gonadotropin (hCG) and its alpha-subunit (hCG-alpha) from the normal human placenta and the effect of some stimulatory agents on their release were studied in vitro using a perfusion system. Each perfusate was assayed for hCG and hCG-alpha in its own homologous radioimmunoassay systems. Both hCG and hCG-alpha were released from the placenta at any stage of gestation in our perfusion system. Much more hCG than hCG-alpha was released from the placenta in early gestation. By comparison, however, hCG-alpha increased gradually with the gestational age. The amount of hCG-alpha released was almost equal to that of hCG in the placenta in the 17th gestational week. After the 22nd gestational week, hCG-alpha was released in larger quantities than hCG, and about 10 times more hCG-alpha than hCG was released from the term placenta. These results were also confirmed by gel filtration of perfusates on a Sephadex G-100 column. hCG-alpha, compared with hCG, was present in excess in gel filtrated perfusates in the last two trimesters. By adding 1 mM dibutyryl cyclic AMP to the perifusion medium, the release of both hCG and hCG-alpha was stimulated significantly. Synthetic luteinizing hormone releasing hormone (LH-RH) at concentrations of 10 ng/ml and 100 ng/ml had no effect, but at a high concentration (1 microgram/ml), LH-RH stimulated the release of them. Moreover, mouse epidermal growth factor (EGF) stimulated not only the release of hCG and hCG-alpha but also their production, because both hCG and hCG-alpha levels rose progressively with the time course in the presence of EGF. The present studies demonstrate that the perifusion system of chorionic tissues is a useful method for investigating the release of hCG and its subunits in vitro.