Potassium iodide (KI) has been shown to have an antigoitrogenic action and to inhibit in vivo thyroid protein biosynthesis. Beef thyroid slices were used to clarify further the mechanism of action of KI. Incubations were performed in Krebs-Ringer-bicarbonate (KRB) buffer under 95%O2 and 5% CO2. KI caused a slight decrease in the uptake of [3H]eucine by the tissue. When labelled leucine incorporation into protein was measured it was found that 10(-6) M KI caused a marked inhibition. Increasing concentrations of KI up to 10(-3) M did not further increase this inhibition. This effect of KI was reduced by simultaneous addition of 0.5 mM KClO4 or 1 mM methylmercaptoimidazole (MMI). In several experiments it was found that equimolar amounts of thyroxine (T4) or triiodothyronine (T3) were more potent than KI in inhibiting thyroid protein biosynthesis. In double plabelled studies KI decreased [3H]leucine incorporation into thyroid soluble proteins and into immunoprecipitable thyroglobulin (T4) while it did not modify that of [14C]galactosamine. When tissue specificity was examined, KI failed to alter [3H]leucine incorporation into proteins either in the liver or in the submaxillary gland. The present results indicate that intracellular KI is necessary to exert its effect on protein synthesis, and that this effect is mediated through a organic form of iodine, probably iodothyronines. This action of KI is specific for the thyroid gland.