We analyzed the TcR Vβ gene usage before and after vaccination with the hepatitis B vaccine since changes in the TcR Vβ gene families would be considered to provide preliminary evidence of a mechanism to prevent HBV infection. Six healthy adult volunteers received immunizations. TcR Vβ usage, T-cell proliferation, and HLA class II alleles were examined in peripheral blood mononuclear cells (PBMC) both before and after vaccination. Furthermore, TcR Vβ usage in postimmunization PBMC was also compared with PBMC cultured with recombinant HBsAg (rHBsAg). The level of in vitro T-cell proliferation in the presence of rHBsAg increased significantly (P < 0.01) in PBMC isolated after vaccinations. Increases in the different TcR Vβ genes were also observed in each individual following vaccinations, regardless of the similarity in their HLA alleles. Specific HBV-related antigen-responsive T cells were induced after HB vaccination, without any common restriction for the TcR Vβ gene families. The mechanism that helps prevent HBV infection was thus found to involve multiclonal alterations in the TcR Vβ repertoire.