The regulation of conversion of thyroxine (T4) to 3,5,3'-triiodothyronine (T3) by the type II iodothyronine deiodinating pathway was studied in normal human placental cells cultured from the chorionic membrane. T4 5'-deiodination was measured in cell sonicates after intact cells were incubated with test agents for 24 to 48 hours. Stimulation of T4 5'-deiodination occurred to a similar degree after depriving cells of thyroid hormone in serum-free medium and in medium containing 10% calf serum. Cortisol at 10 to 100 nmol/L in serum-free medium inhibited T4 5'-deiodination up to 36%, and 1 to 100 nmol/L of insulin inhibited deiodination up to 50%. Dibutyryl-cyclic AMP (dbcAMP) inhibited deiodination, but this appeared to result from the inhibitory effects of butyrate. Addition to the culture media of 8-bromo-cAMP, cholera toxin, and theophylline each caused partial inhibition of T4 5'-deiodination, strongly suggesting an inhibitory effect of raised intracellular cAMP. Neither alpha- nor beta-adrenergic agonists had any effect when added to the culture medium, nor did glucagon or cysteamine. These results demonstrate a complex, multihormonal control of human placental type II iodothyronine deiodination, and suggest that changes in the activity of this pathway may result in altered intracellular, and conceivably circulating, T3 concentrations in states of cortisol excess and marked hyperinsulinism. The factor that regulates type II deiodination via cAMP remains to be identified.