Following appropriate antigen-specific stimulation, CD4(+) and CD8(+) T lymphocytes rapidly express cytokines. Based on this stimulation-induced cytokine secretion and using cell surface 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.