The time sequence of radioiodine sequestration and secretion (BE131I) have been compared in dog thyroid slices prelabeled with 131I in vivo and incubated in vitro with or without TSH. Sequestration has been taken to be the amount of radioiodine present in phagocytic vacuoles or colloid droplets; the TSH or (Bu)2cAMP stimulation of the basal values was suppressed by endocytosis blocking drugs. TSH induced a sequestrated radioactivity (S) after 5 min and a stimulated secretion after 20 min. The secretion rate was constant: 1%/h (mean +/- SD = 1.0 +/- 0.4; n = 7) of the total radioactivity of the slices. At equilibrium, S was constant and equal to less than 1% of the total radioactivity. The half-life of S, assuming a disappearance rate proportional to S, was 26 min (26 +/- 4; n = 5); assuming a disappearance rate independent of S, the lifetime was 44 min (44 +/- 7; n = 6). At the steady state, the limiting step of maximally stimulated secretion was the hydrolysis of the sequestrated radioactivity and endocytosis rate was equal to secretion rate. Without TSH, a constant BEI release (0.23% +/- 0.07%/h; n = 7), insensitive to cytochalasin B, was observed, which corresponded to basal secretion.