The pore of the translocon complex in the endoplasmic reticulum (ER) is large enough to be permeated by small molecules, but it is generally believed that permeation is prevented by a barrier at the luminal end of the pore. We tested the hypothesis that 4-methylumbelliferyl alpha-d-glucopyranoside (4MalphaG), a small, neutral dye molecule, cannot permeate an empty translocon pore by measuring its activation by an ER resident alpha-glucosidase, which is dependent on entry into the ER. The basal entry of dye into the ER of broken Chinese hamster ovary-S cells was remarkably high, and it was increased by the addition of puromycin, which purges translocon pores of nascent polypeptides, creating additional empty pores. The basal and puromycin-dependent entries of 4MalphaG were mediated by a common, salt-sensitive pathway that was partially blocked by spermine. A similar activation of 4MalphaG was observed in nystatin-perforated cells, indicating that the entry of 4MalphaG into the ER did not result simply from the loss of cytosolic factors in broken cells. We reject the hypothesis and conclude that a small, neutral molecule can permeate the empty pore of a translocon complex, and we propose that translationally inactive, ribosome-bound translocons could provide a pathway for small molecules to cross the ER membrane.