Different HLA class I alleles display a distinctive dependence on tapasin for surface expression and Ag presentation. In this study, we show that the tapasin dependence of HLA class I alleles correlates to the nature of the amino acid residues present at the naturally polymorphic position 114. The tapasin dependence of HLA class I alleles bearing different residues at position 114 decreases in the order of acidity, with high tapasin dependence for acidic amino acids (aspartic acid and glutamic acid), moderate dependence for neutral amino acids (asparagine and glutamine), and low dependence for basic amino acids (histidine and arginine). A glutamic acid to histidine substitution at position 114 allows the otherwise tapasin-dependent HLA-B4402 alleles to load high-affinity peptides independently of tapasin and to have surface expression levels comparable to the levels seen in the presence of tapasin. The opposite substitution, histidine to glutamic acid at position 114, is sufficient to change the HLA-B2705 allele from the tapasin-independent to the tapasin-dependent phenotype. Furthermore, analysis of point mutants at position 114 reveals that tapasin plays a principal role in transforming the peptide-binding groove into a high-affinity, peptide-receptive conformation. The natural polymorphisms in HLA class I H chains that selectively affect tapasin-dependent peptide loading provide insights into the functional interaction of tapasin with MHC class I molecules.