Human leukocyte antigen (HLA) class I molecules are critical components of the cell-mediated immune system that bind and present intracellular antigenic peptides to CD8(+) T cell receptors. To understand the interaction mechanism underlying human leukocyte antigen (HLA) class I specificity in detail, we studied the structural interaction characteristics of 16,393 nonameric peptides binding to 58 HLA-A and -B molecules. Our analysis showed for the first time that HLA-peptide intermolecular bonding patterns vary among different alleles and may be grouped in a superfamily dependent manner. Through the use of these HLA class I 'fingerprints', a high resolution HLA class I superfamily classification schema was developed. This classification is capable of separating HLA alleles into well resolved, non-overlapping clusters, which is consistent with known HLA superfamily definitions. Such structural interaction approach serves as an excellent alternative to the traditional methods of HLA superfamily definitions that use peptide binding motifs or receptor information, and will help identify appropriate antigens suitable for broad-based subunit vaccine design.