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T cell receptors, which recognize antigen peptides on MHC molecules, are essential probes for the analysis of T cell antigen specificity. The identification of paired T cell receptor (TCR) chains, α/β or γ/δ, usually requires the establishment of T cell clones, which is not always available. In this study, we tried, as an alternative method, the paired cloning of TCR α/β genes directly from a single T cell. T cells were sorted as a single cell from which RNA was extracted. Then, TCR α/β CDR3 regions were amplified from the single cell-derived cDNA by reverse transcriptase-polymerase chain reaction to determine their sequences. We successfully identified pairs of TCR α/β genes, and reconstructed the TCR molecule by a bacterial expression system. This strategy makes it possible to obtain recombinant TCR molecules from a single T cell without cellular cloning and promotes the investigation of T cell antigen specificity.