The uses of soluble HLA class I/peptide complexes to monitor antigen reactive T cells are often hampered by their low-yield and high-cost production. As an alternative strategy, the peptide-beta(2)m fused, 2-component (2C) HLA class I/peptide complex has been developed, but its application is limited due to the lack of the comparison of its structural and functional characteristics with those of its conventional 3-component (3C) counterpart. In this study, we have demonstrated that the 2C and 3C HLA-A2/MART1(27-35) complexes have a similar chromatographical profile and comparable stability, but the former has 2.5 times higher yield and significantly higher binding ability with HLA-A2/MART1(27-35) complex-specific receptors than the latter. Furthermore, the 2C complex has a comparable ability to stimulate specific CTL proliferation, but appears to be more effective in eliciting the cytotoxicity of antigen-specific CTL, as compared to its 3C counterpart.