In the cultured human hepatoma HepG2, Ca2+ ionophores block secretion of different secretary proteins to different extents, alpha 1-antitrypsin secretion being more sensitive to A23187 and ionomycin than is alpha 1-antichymotrypsin, and albumin secretion the least of the three proteins studied. As judged by subcellular fractionation experiments and by treatment of pulse chase labeled protein with endoglycosidase H, A23187 and ionomycin cause newly made secretory proteins to remain within the rough endoplasmic reticulum (ER). Experiments in which A23187 is added at different times during a pulse or chase show that secretion of newly made alpha 1-antitrypsin becomes resistant to the ionophore, on average, 15 min after synthesis; this is about 20 min before it reaches the trans-Golgi, and while it is still within the rough ER. We speculate that a high concentration of Ca2+ within the ER may be essential for certain secretory proteins to fold properly, that folding is inhibited when ER Ca2+ levels are lowered by ionophore treatment, and that unfolded proteins, particularly alpha 1-antitrypsin, cannot exit the rough ER. Treatment of murine 3T3 fibroblasts or human hepatoma HepG2 cells with the Ca2+ ionophores A23187 or ionomycin also induces a severalfold accumulation of the ER lumenal protein Bip (Grp78). These findings disagree with a recent report that Ca2+ ionophores cause secretion of Bip and other resident ER proteins, but is consistent with other reports that A23187 causes accumulation of mRNAs for Bip and other ER lumenal proteins.