In order to improve the processing efficiency of T cell tumor antigen epitopes, this bioinformatic study compares proteolytic sites in the generation of 47 experimentally identified HLA-A2.1-restricted immunodominant tumor antigen epitopes to those of 52 documented HLA-A2.1-restricted immunodominant viral antigen epitopes. Our results show that the amino acid frequencies in the C-terminal cleavage sites of the tumor antigen epitopes, as well as several positions within the 10 amino acid (aa) flanking regions, are significantly different from those of the viral antigen epitopes. In the 9 amino acid epitope region, frequencies differed somewhat in the secondary-anchored amino acid residues on E3 (the third aa of the epitope), E4, E6, E7 and E8; however, frequencies in the primary-anchored positions, on E2 and E9, for binding in the HLA-A2.1 groove, remained almost identical. The most frequently occurring amino acid pairs in both N-terminal and C-terminal cleavage sites in the generation of tumor antigen epitopes were different from those of the viral antigen epitopes. Our findings demonstrate for the first time that these two groups of epitopes may be cleaved by distinct sets of proteasomes and peptidases or similar enzymes with lower efficiencies for tumor epitopes. In the future, in order to more effectively generate tumor antigen epitopes, targeted activation of the immunoproteasomes and peptidases that mediate the cleavage of viral epitopes could be achieved, thus enhancing our potential for antigen-specific tumor immunotherapy.