Calreticulin (CRT) is an Endoplasmic Reticulum (ER) resident protein involved in intracellular calcium homeostasis and the folding of newly synthesised glycoproteins. CRT also forms part of the peptide loading complex (PLC) where it assists in Major Histocompatibility Complex (MHC) class I folding, maturation and peptide loading with the assistance of ERp57. CRT's localisation is kept in check through a C-terminal Lys-Asp-Glu-Leu (KDEL) sequence that retrieves it to the ER through the KDEL receptor. The CRT -/- mouse fibroblast cell line K42 loads MHC class I molecules and presents endogenous antigens to cytotoxic T lymphocytes inefficiently ~Gao et al 2002). Importantly, the trafficking speed of the endogenous class I alleles (H-2Kb and H-2D ) to the cell surface was shown to be faster in K42 than in its CRT +/+ counterpart K4I; furthermore fewer MHC I were incorporated into the PLC in K42 (Gao et aI., 2002), similar to a class I Tl34K mutant that fails to present antigen efficiently (Lewis et aI., 1996). This suggests that CRT influences MHC I trafficking rate and may recruit MHC I to the PLC, ensuring efficient MHC I antigen presentation. In this study, the role of CRT was further dissected using a series of mutant constructs that were expressed in K42 and their effects on MHC I antigen presentation determined. The mutants were designed to address three questions: the role of the c-terminus and KDEL in MHC I retention / retrieval; the importance of the interaction between CRT and ERp57; and the effect of mutation of CRT's glycan-binding site on MHC I antigen presentation. Unexpectedly, the glycan-binding CRT mutant fully restored MHC I antigen presentation, suggesting that polypeptide interactions define CRT substrate specificity. However, efficient MHC I assembly with the PLC was shown to require an interaction between CRT and ERp57. The trafficking rate of MHC I was influenced not just by the KDEL sequence but also by the interaction between CRT and ERp57 and by undefined residues within the 11 c-terminal amino acids of CRT, consistent with a role in MHC I retention / retrieval. Cumulatively, results show that the CRT's c-terminus, KDEL sequence and ERp57 binding site, but not CRT's glycan-binding site, are obligatory for efficient MHC I antigen presentation.