CDR3 of the heavy-chain variable region of immunoglobulin is a region in which somatic mutation occurs heavily after secondary antibody response, resulting in an affinity maturation of antibodies in vivo. The aim of this study was to improve the affinity of a human single-chain variable fragment (scFv) specific for pre-S1 of hepatitis B virus (HBV) by introducing random mutagenesis in CDR3 variable region of heavy chain (V(H)) of the parental scFv clone 1E4. By using a BIAcore for panning and screening, we have selected three clones (A9, B2, and B9) with lower highest affinity (K(D)) than 1E4. Affinities of selected clones ranged from 1.7 x 10(7) mol/L to 6.3 x 10(8) mol/L, which were increased by factors of 1.4 to 4.0, respectively, compared to the parental clone. Binding inhibition assay using flow cytometry and polymerase chain reaction revealed that B2 (6.4 x 10(8) mol/L) had a higher neutralizing activity against pre-S1 or HBV virion binding to liver cell line. This anti-pre-S1 scFv can be considered as a potential therapeutic tool for a passive immunotherapy for HBV infection.