Perforin- and Fas-based cytolytic pathways are two major mechanisms of cell-mediated cytotoxicity. Recently, we have shown that an inhibitor of vacuolar type H+-ATPase, concanamycin A (CMA), inhibits perforin-based cytotoxic activity, mostly due to accelerated degradation of perforin by an increase in the pH of lytic granules. Here we show that CMA failed to inhibit the cytolytic activity of CD4+ CTL clone and perforin-deficient CD8+ CTL clone, which exclusively mediate Fas-based cytotoxicity, although CMA inhibited acidification and induced drastic vacuolation of cytoplasmic granules in these clones. In a wide range of alloantigen-specific CTL, a significant amount of the lysis of Con A blasts from normal mice and of Fas-positive tumor cells remained unaffected even in excess concentrations of CMA. However, CMA almost completely inhibited the lysis of Con A blasts from lpr mice and of Fas low expressing or negative tumor cells. Cytolysis by alloantigen-specific CD8+ CTL derived from gld mice was completely prevented by CMA. Furthermore, CMA-insensitive cytolysis exerted by CD8+ CTL clone was completely inhibitable by soluble Fas molecules. Thus, these data clearly indicate not only that CMA-insensitive cytolysis mediated by alloantigen-specific CTL is Fas dependent, but also that CMA is a selective inhibitor to block only the perforin-based killing pathway. In contrast, brefeldin A blocked the Fas-based cytotoxicity, but only marginally reduced the perforin-based cytotoxicity. Moreover, CMA and brefeldin A in combination completely abrogated all cytolytic activity of alloantigen-specific CTL. Taken together, these results reveal that CTL mainly exert perforin-based cytotoxicity and complementary Fas-based cytotoxicity, and that CMA is a powerful tool to clarify the contributions of the two distinct cytolytic pathways.