The low density lipoprotein receptor-related protein (LRP) is a cysteine-rich, multifunctional receptor that binds and endocytoses a diverse array of ligands. Recent studies have shown that a 39-kDa receptor-associated protein (RAP) facilitates the proper folding and subsequent trafficking of LRP within the early secretory pathway. In the current study, we have examined the potential role of Ca2+ and its relationship to RAP during LRP folding. We found that depletion of Ca2+ following either ionomycin or thapsigargin treatment significantly disrupts the folding process of LRP. The misfolded LRP molecules migrate as high molecular weight aggregates under nonreducing SDS-polyacrylamide gel electrophoresis, suggesting the formation of intermolecular disulfide bonds. This misfolding is reversible because misfolded LRP can be re-folded into functional receptor molecules upon Ca2+ restoration. Using an LRP minireceptor representing the fourth ligand binding domain of LRP, we also observed significant variation in the conformation of monomeric receptor upon Ca2+ depletion. The role of Ca2+ in LRP folding is independent from that of RAP because RAP remains bound to LRP and its minireceptor following Ca2+ depletion. Furthermore, Ca2+ depletion-induced LRP misfolding occurs in RAP-deficient cells. Taken together, these results clearly demonstrate that Ca2+ and RAP independently participate in LRP folding.