The cytotoxic activity and pore-forming protein (PFP) expression of human peripheral blood (PB) gamma/delta T cells were examined. Fresh gamma/delta T cells isolated from PB lymphocytes by fluorescence-activated cell sorting exhibited a substantial natural killer-like cytotoxic activity against K562 target cells and had a high cytotoxic potential triggered by anti-CD3 monoclonal antibody (mAb) against P815 target cells bearing Fc gamma R. Immunocytochemical staining with an anti-PFP mAb revealed that virtually all PB gamma/delta T cells are granular lymphocytes with abundant PFP in their cytoplasmic granules. Constitutive expression of PFP in PB gamma/delta T cells was also demonstrated by Northern blot analysis. These observations support the proposed role of gamma/delta T cells in cytolytic immune surveillance in vivo.