Following appropriate antigen‐specific stimulation, CD4⁺ and CD8⁺ T lymphocytes rapidly express cytokines. Based on this stimulation‐induced cytokine secretion and using cell suface affinity matrix technology we have developed a new method that permits specific, rapid and efficient detection, isolation and characterization of live antigen‐specific CD4⁺ and CD8⁺ T lymphocytes. The power of this technique is demonstrated here for HLA‐A0201‐restricted influenza matrix protein peptide 58‐66‐specific CD8⁺ cytotoxic T lymphocytes, influenza A virus‐ and recombinant tetanus toxin C fragment‐specific Th1 cells and tetanus toxoid‐specific Th2 cells.