We have previously detected perforin expression in a subpopulation of asialo GM1+ natural killer (NK) cells and CD8+ T lymphocytes in murine spleen cells by immunocytochemical staining with an anti-perforin monoclonal antibody. In the present study, more detailed analyses of perforin expression in murine cytotoxic lymphocyte subpopulations were performed. The expression of perforin in asialo GM1+ spleen cells was predominantly confined to the NK1.1+ subset, where all NK activity also resided. Perforin expression was also studied on alloreactive cytotoxic T lymphocyte (CTL) induced in vivo. The cells expressing perforin in peritoneal exudate lymphocytes predominantly resided in the CD8+ T cell subpopulation co-expressing asialo GM1 where an allospecific CTL activity also resided. Furthermore, the percentage of perforin-positive cells in this population was greatly reduced after stimulation with anti-CD3 or anti-T cell receptors antibodies, which induce serine esterase release from the cytoplasmic granules. These findings highly suggest that perforin is involved in in vivo NK cell- and CTL-mediated cytolysis.