Treatment of FRTL-5 cells with a Ca2+ ionophore, A23187, or a specific inhibitor of the endoplasmic reticulum Ca2+ ATPases, thapsigargin, delayed thyroglobulin secretion. The secreted thyroglobulin showed an increased electrophoretic mobility and a reduced sensitivity to neuraminidase. Only thyroglobulin that was still in the endoplasmic reticulum was sensitive to the Ca(2+)-perturbant drugs as shown by experiments in which the drugs were added at different times during a chase. Analysis of the carbohydrate chains by BioGel P4 showed that thyroglobulin secreted in the presence of the Ca(2+)-perturbants displayed an increased ratio high mannose/complex chains.