Isolating antigen-specific T lymphocytes is hampered by the low frequency of the cells and the low affinity between T-cell receptors (TCR) and antigen. We describe the isolation and purification of antigen-specific CD8⁺ T lymphocytes from mixed T-cell populations. Magnetic beads coated with major histocompatibility complex class I molecules loaded with specific peptide were used as a substrate for T-cell capture. Low-frequency T cells, as well as T cells with TCR of low affinity for the antigen were captured on the beads. Following isolation and expansion, recovered cells specifically killed target cells in vitro, and displayed antiviral effect in vivo.