Many viral epitope specific T cell receptors (TCRs) in MHC-matched individuals have been demonstrated to involve conserved amino acid motifs in β chain complementarity-determining region 3 (CDR3). However, it is notsure whether the conserved motifs can also be found in TCR α chain. In previous studies, we developed a modified method to enlarge the percentage of cytomegalovirus (CMV) pp65 peptide-specific CD8+ T cells in PBMC by continuous peptide stimulation in vitro, which provides sufficient number of specific T cells for detection. In this study, we further analyzed the restrictive usage of TCR Vα and Vβ gene families and investigated the CDR3 gene sequence of pp65 peptide-specific CD8+ T cells. Analysis of CDR3 spectratypes suggested a restricted usage of TCR α chain AV8, AV12, AV21, AV31 families and TCR β chain BV3, BV14, BV21, BV23, BV11 families in donor CD8+ T cells stimulated by pp65 peptide. The sequences of these T cells involved similar sequence (TX) G (X) A in CDR3 region of TCR α chain and L (XT) G (X) A in TCR β chain. Cellular & Molecular Immunology.