The effect of varying the concentration of Ca++ and Mg++ ions of the medium on in vitro thyroidal 131I release was studied. There was no effect when Ca++ or Mg++ was removed from the medium, but elevated levels of either divalent cation inhibited both TSH- and cyclic AMP-stimulated 131I release. Preincubation in Ca++-Mg++-free medium only slightly inhibited the release of 131I. Addition of citrate or EGTA to the medium led to release of iodoprotein without a distinct inhibition of the TSH response. TSH, however, inhibited the chelator-induced iodoprotein release. It is suggested that TSH releases stored intracellular Ca++ and this may play a role in the activation of secretion. (Endocrinology90: 1459, 1972)