The authors previously established, using modeling studies, the residues in calreticulin (CRT) important for sugar binding. Here, the authors discuss the relative roles of CRT residues Trp-319, Asp-317, and Asp-160 for sugar binding by using site-directed mutagenesis and isothermal titrn. calorimetry (ITC). Residues corresponding to Asp-160 and Asp-317 in calnexin (CNX) also play important roles in sugar binding. From the present study, the authors demonstrated that residue Asp-160 was not involved in sugar binding, while Asp-317 plays a crucial role. Further, it was also validated that cation-p interactions of the sugar with Trp-319 dictated sugar binding in CRT. This study not only further defined the binding site of CRT but also highlighted its subtle differences with that of CNX. [on SciFinder (R)]