To assess the importance of each amino acid residues in HVEM(23–39) interaction with BTLA protein, single alanine-substituted HVEM(23–39) variants were synthesized [38, 39] and tested in the BTLA/HVEM ELISA. While no or minor diminution in the inhibition of BTLA/HVEM binding was found with the majority of alanine-substituted fragments compared to the native HVEM(23–39), we observed a significant reduction of the inhibition, in a dose depend manner, for the two peptides where cysteine in position 29 (C29A) or 37 (C37A) was replaced by an alanine (Fig 4). This suggests the possible importance of cysteine residues in inhibition BTLA/HVEM complex formation by the HVEM(23–39) peptide.