- We investigated the requirement for calnexin in the biogenesis of MHC class I molecules. Mutant human cells lacking calnexin were infected with recombinant vaccinia viruses encoding mouse MHC class I molecules, K d , Kb , Kk , D d , Db , and Ld . Flow cytometry indicated that each of the six MHC class I allomorphs was transported to the cell surface at similar rates in calnexin-deficient cells and transfectants expressing calnexin. For Kb and K d , the calnexin-independent biogenesis occurred regardless of whether the MHC class I molecules contained human or mouse β2-microglobulin. Also addressed was the effect of calnexin on the surface expression of Kb molecules bearing the immunodominant peptide from ovalbumin (OVA257 – 264 ). This was detected with a recently described monoclonal antibody specific for the Kb/peptide complex. Calnexin expression had no significant effect on the formation of Kb /peptide complexes generated from full-length OVA, cytosolic OVA257 – 264 , or endoplasmic reticulum-targeted OVA257 – 264 , which was expressed in the presence of the herpes simplex virus ICP47 protein to ensure detection of TAP-independent peptide-MHC class I complexes. Complementary results were obtained with TAP-independent formation of K d /peptide complexes. These findings indicate that calnexin is not required for the efficient assembly of MHC class I molecules with TAP-dependent or independent peptides.