- After brief incubation of cells with fluorescein-conjugated peptides that bind major histocompatibility complex (MHC) class I molecules, peptides were detected within the endoplasmic reticulum (ER) by microscopy or by binding to radiolabeled class I molecules. ER delivery of a nonfluorescent peptide was demonstrated using a mAb highly specific for the peptide-class I molecule complex. ER localization of peptides: (i) required expression of appropriate class I molecules in the ER but not on the cell surface, (ii) was diminished by expression of TAP, the MHC-encoded cytosol to ER peptide transporter, and (iii) was blocked by pinocytosis inhibitors but not by brefeldin A. These findings demonstrate the existence of a pathway, likely vesicular in nature, that conveys small extracellular substances to the ER without traversing the Golgi complex or the cytosol. This pathway contributes to the loading of exogenous peptides to MHC class I molecules, but its evolutionary significance may lie in other cellular processes, such as maintaining ER homeostasis or signaling by extracellular substances.