Wild-type human chorionic gonadotropin (hCG) has been used as a contraceptive vaccine. However, extensive sequence homology with LH (LH) elicits production of cross-reactive antibodies. Substitution of arginine⁶⁸ of the -subunit (hCG) with glutamic acid (R68E) profoundly reduces the cross-reactivity while refocusing the immune response to the hCG-specific C-terminal peptide (CTP). To investigate the molecular basis for this change in epitope usage, we immunized mice with a plasmid encoding a truncated hCG-R68E chain lacking the CTP. The animals produced LH-cross-reactive antibodies suggesting that the refocused immunogenicity of R68E is a consequence of epitope masking by a novel disposition of the CTP in the mutant rather than a structural change in the cross-reactive epitope region. This explanation was strongly supported by surface plasmon resonance analysis using a panel of anti-hCG-specific and anti-hCG/LH cross-reactive monoclonal antibodies (mAbs). While the binding of the LH cross-reactive mAbs to hCG-R68E was eliminated, mAbs reacting with hCG-specific epitopes bound to hCG and hCG-R68E with identical affinities. In a separate series of experiments, we observed that LH cross-reactive epitopes were silent following immunization with a plasmid encoding a membrane form of hCG-R68E, as previously observed with the soluble mutant protein itself. In contrast, the plasmid encoding the soluble secreted form of hCG-R68E evoked LH cross-reactive antibodies, albeit of relatively low titer suggesting that the handling and processing of the proteins produced by the two constructs differed.