An inhibitor of vacuolar type H(+)-ATPase, concanamycin A (CMA), inhibited the specific cytolytic activity of a CD8+ CTL clone, OE4. The inhibitory effect was observed when the effector cells, and not the target cells, were pretreated with CMA. CMA did not seem to inhibit early events, inasmuch as effector/target conjugate formation remained unaffected. Although CMA treatment of OE4 resulted in a slight decrease in the efficiency of granule exocytosis in response to anti-CD3 stimulation, the most prominent effect was a marked reduction of perforin activity and DNA degradation activity in lytic granules. Western blotting analysis indicated a drastic decrease in the amount of perforin in CMA-treated cells. Fluorescent microscopic observation of OE4 stained with acridine orange indicated that CMA raised the pH of the lytic granules. Under transmission electron microscopy, striking morphologic changes in cytoplasmic granular structures were observed after CMA treatment of OE4. The lytic granules of OE4 had homogeneously stained large cores and numerous small vesicles that filled peripheral areas. In contrast, the lytic granules of CMA-treated OE4 showed irregular shapes with no small vesicles, but with cores that became rough and loose. Vacuoles with no structure in them were seen occasionally. These results suggest that acidification through vacuolar type H(+)-ATPase is essential to maintain the structure and function of lytic granules.