Local immunosuppression may explain the failure of an effective immune response against solid tumors. Although it is well known that the interstitial pH is significantly lower in solid tumors than in normal tissue, only a few studies in the mouse system have investigated the influence of this acidic milieu on the anti-tumoral cytotoxic response. Here the authors report the suppression of human non-major histocompatibility complex (MHC)-restricted cytotoxicity against tumor cells by an acidic extracellular pH (pHe). Unstimulated peripheral blood mononuclear cells, lymphokine-activated killer (LAK) cells, and natural killer cell clones were used as effector cells. According to pH measurements in solid tumors, representative pH values of 7.2 to 5.3 were chosen during the cytotoxic assays. Target cell lysis was measured using two nonradioactive fluorometric methods, namely two-color flow cytometry and a modified calcein-release assay, which allowed cell-mediated cytotoxicity to be measured and compared with that in adherent targets. Using K562, Daudi, or Raji as suspended target cell lines, the cytotoxic activity of unstimulated peripheral blood mononuclear cells and of LAK cells was markedly reduced by a decreasing pHe. An extracellular pH of 5.8 to 5.3 resulted in a nearly complete loss of the cytotoxic response. This pHe-dependent impairment of the killing activity could also be shown for killer cells stimulated with interleukins-7 and -12, phytohemagglutinin, or lipopolysaccharide. The lytic potential of homogeneous natural killer cell clones as effectors was also strictly influenced by the surrounding pH. The pHe dependence of the non-MHC-restricted killer cell functions against tumor cells seems to be a general phenomenon, because the cytolytic activity of LAK cells against six human adherent tumor cell lines (HeLa, HepG2, LS174T, LS174Te, MCF-7, and RT112) was also clearly reduced under acidic conditions. To initiate the killing process, adhesion molecules play an important role in recognition and binding of the target cell. However, flow cytometric analysis revealed that the expression pattern of relevant adhesion molecules was unaffected by acidic pHe. In conclusion, these data clearly indicate an inhibition of non-MHC-restricted cytotoxicity against tumor cells by an acidic pHe, which may contribute to the failure of immunosurveillance against solid tumors. Consequently, efforts to enhance the anti-tumoral cytotoxicity by immunotherapies may have limited success.