Significance A newly discovered system for immunological detection of diverse bacterial and fungal pathogens involves the MHC-like host protein called MR1. This molecule scavenges metabolites from the biosynthesis of riboflavin by microbes. MR1 presents these compounds on the surface of antigen-presenting cells, where they interact with T cells known as mucosal-associated invariant T cells and stimulate immunity. Critical aspects of the cell biology of metabolite presentation by MR1 are unknown. Here we generated a fluorescent antigen analog and use it to show that MR1 captures its metabolites within the endoplasmic reticulum. We describe proteins that maintain MR1 ready for metabolite binding in the endoplasmic reticulum to promote efficient pathogen detection. MR1 thus monitors extracellular microbial metabolites from within the cell.