The effect of TSH, phorbol ester, norepinephrine (NE), and carbachol, agents known to influence thyroid metabolism, was compared on iodide organification in dog thyroid slices, freshly isolated follicles, and cultured cells. TSH stimulated iodide organification in all three types of preparations, and this effect was mimicked by (Bu)2cAMP. In contrast, the phorbol ester, tetradecanoyl phorbol acetate (TPA) stimulated iodide organification in slices and follicles but inhibited it in cells. The dose and time required for these divergent effects were similar. Other stimulators of protein kinase C such as aplysiatoxin and teleocidin mimicked the effects of TPA, and these effects were partially reversed by H-7, an inhibitor of protein kinase C. Pretreatment of cells with TPA for 4 h did not affect TSH-stimulated cAMP production, but TPA inhibited iodide organification in cells even in the presence of TSH or (Bu)2cAMP. Similarly NE and carbachol stimulated iodide organification in follicles but inhibited it in cells under basal as well as TSH-stimulated conditions. These effects of NE and carbachol were via alpha 2-adrenergic and muscarinic cholinergic receptors, respectively. However, NE and carbachol inhibited TSH-stimulated cAMP production in both follicles and cells. In thyroid cells, carbachol inhibited uptake and increased efflux of iodide within minutes. TPA also produced similar effects after longer periods of incubation, where an inhibition of uptake was seen by 1 h and an increase in efflux by 2 h. NE had a marginal inhibitory effect on uptake and had no effect on efflux of iodide. In contrast to these agents, TSH increased uptake but not efflux of iodide. The present results suggest that the response of the freshly isolated tissue to phorbol esters, NE and carbachol differs from that of cells in culture with respect to an important metabolic function of the thyroid gland. These agents seem to have direct effects on iodide transport and organification unrelated to their effects on cAMP production.