Human chorionic gonadotropin (hCG) secretion was studied in human malignant trophoblast cells (Jar line) in continuous culture. Radioimmunoassayable hCG in the cells and in the daily culture fluid was increased for at least 3 days by 10% newborn calf serum and by 1 mM N6, 02′-dibutyryl cyclic adenosine 3′,5′-monophosphoric acid (dbcAMP) plus 1 mM theophylline. Incubation of the cells in the presence of serum, dbcAMP and theophylline resulted in additive stimulation of hCG secretion after 1 day. After 2 or 3 days, a supra-additive stimulation was observed, which was prevented by Actinomycin D. When cells were preincubated for 2 days in the absence of serum but with dbcAMP plus theophylline, subsequent addition of serum resulted in supra-additive stimulation of hCG secretion within a single day; the continued presence of dbcAMP plus theophylline was not necessary for this stimulation by serum. These findings suggested that dbcAMP activates a slow process requiring RNA synthesis, resulting in increased hCG secretion. Serum appears to protect the hCG synthesizing system from degradation.