We have examined the effects of depleting the endoplasmic reticulum Ca2+ store on the maturation of newly synthesized thyroglobulin molecules, their export to the Golgi complex, and their secretion by FRTL-5 cells. An inhibitor of the endoplasmic reticulum Ca2+ pump, thapsigargin, and the Ca2+ ionophore A23187 depleted the endoplasmic reticulum Ca2+ store and strongly inhibited thyroglobulin secretion in cells chased in medium containing 0.1 mM Ca2+. Inhibition of thyroglobulin secretion was caused by a block in the export of newly synthesized thyroglobulin molecules from the endoplasmic reticulum to the Golgi complex, as shown by cell-fractionation experiments and the intracellular accumulation of endoH-sensitive thyroglobulin. The thyroglobulin molecules retained in the endoplasmic reticulum of cells treated with the drugs were found to assemble more slowly into dimers than thyroglobulin in control cells. Protease-sensitivity experiments demonstrated that thyroglobulin dimers assembled in the presence of thapsigargin had a different conformation with respect to dimers assembled in controls cells.