To verify the physiological role of the pore-forming protein perforin in vivo, its expression in subpopulations of human peripheral blood lymphocytes was examined by immunocytochemical staining and their cytolytic potentials compared. In addition to NK cells and gamma delta T cells, which uniformly expressed abundant perforin in their cytoplasmic granules, only a small subpopulation of CD8+ alpha beta T cells contained perforin, namely the CD11b+ subset. However, in vitro activation with an anti-CD3 antibody and IL-2 induced perforin expression in approximately 50% of the CD8+CD11b- T cells and also in a small subset of CD4+ T cells. A distribution of perforin in CD8+ and CD4+ T cells, similar to in vitro activated T cells, was observed in fresh peripheral blood lymphocytes from infectious mononucleosis patients. In all instances, the expression of perforin correlated with the cytolytic potential of these subpopulations. The results strongly suggest that perforin plays a role in the manifestation of cytotoxic activity in vivo.