Human malignant trophoblast cells (BeWo line) in culture were employed to investigate the early stimulation of human chorionic gonadotropin (hCG) secretion by 1 mM dibutyryl cyclic AMP and 1 mM theophylline (dbT). The earliest increase in secreted immunoreactive hCG occurred at 3 1/2 h following addition of dbT, and was preceded by an increase in intracellular hCG. These results suggested that dbT stimulated hCG synthesis, rather than release. Addition of either 0.062 mug/ml actinomycin D or 100 mug/ml cordycepin along with dbT, or 3 mug/ml alpha-amanitin 9 h prior to addition of dbT, prevented the increase in hCG secretion at 3 1/2-8 h. It was concluded that synthesis of RNA (possibly messenger RNA) is required for the early stimulation of hCG secretion by dbT.