For the site-directed conjugation of chemicals and radioisotopes to the chicken-derived single-chain variable fragment (scFv), we investigated amino acid residues replaceable with cysteine. By replacing each amino acid of the 157 chicken variable region framework residues (FR, 82 residues on VH and 75 on VL) with cysteine, 157 artificial cysteine mutants were generated and characterized. At least 27 residues on VL and 37 on VH could be replaced with cysteine while retaining the binding activity of the original scFv. We prepared three VL (L5, L6 and L7) and two VH (H13 and H16) mutants as scFv-Ckappa fusion proteins and showed that PEG-conjugation to the sulfhydryl group of the artificial cysteine was achievable in all five mutants. Because the charge around the cysteine residue affects the in vivo stability of thiol-maleimide conjugation, we prepared 16 charge-variant artificial cysteine mutants by replacing the flanking residues of H13 with charged amino acids and determined that the binding activity was not affected in any of the mutants except one. We prepared four charge-variant H13 artificial cysteine mutants (RCK, DCE, ECD and ECE) as scFv-Ckappa fusion proteins and confirmed that the reactivity of the sulfhydryl group on cysteine is active and their binding activity is retained after the conjugation process.