The endoplasmic reticulum (ER) is the site for assembly of MHC class I molecules. Newly synthesized class I heavy chains bind calnexin, an ER-resident molecular chaperone, and dissociate from calnexin following association with beta2-microglobulin (beta2m). The class I heavy chain:beta2m complex then is stabilized by binding endogenous peptides transported to the ER through the TAP molecules. Thus, both beta2m and TAP are required for class I Ag presentation. Human CD1b is a beta2m-associated, non-MHC-encoded glycoprotein that functions in presentation of lipid Ags to T cells. Despite its structural similarities with class I, CD1b-mediated Ag presentation is TAP independent, and CD1b traffics to endocytic compartments for sampling exogenous Ags. Given these distinctive features of CD1b, we set out to analyze its assembly. Immunofluorescence microscopic analysis of CD1b-transfected cells with a rabbit heteroantiserum detecting only beta2m-free CD1b heavy chains revealed a reticular pattern characteristic of ER staining. Consistently, CD1b heavy chains recognized by this antiserum were sensitive to endoglycosidase H and associated with calnexin, indicating a distribution restricted to the ER. Furthermore, CD1b heavy chains were confined to the ER in beta2m-negative cells, whereas they exited the ER and distributed in post-Golgi compartments when beta2m was reconstituted in these cells. These results suggested that assembly of CD1b heavy chains with beta2m occurred in the ER and that only assembled CD1b heavy chain:beta2m complexes were able to exit the ER and traffic to endocytic compartments, where they encounter endocytosed lipid Ags.