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The dependence of sulphur mustard (HD) toxicity on intracellular (pHi) and extracellular pH was examined in CHO-K1 cells. HD produced an immediate and significant concentration-dependent decline in cytosolic pH, and also inhibited the mechanisms responsible for restoring pHi to physiological values. The concentration–response of HD-induced cytosolic acidification, closely paralleled the acidification of the extracellular buffer through HD hydrolysis. A viability study was carried out in order to assess the importance of HD-induced cytosolic acidification. Cultures were exposed to HD for 1 h in media that were adjusted through a pH range (pH 5.0–10), and the 24 h LC50 values were assessed using the viability indicator dye alamarBlue™. The toxicity of HD was found to be dependent on extracellular pH, with a greater than eight-fold increase in LD50 obtained in cultures treated with HD at pH 9.5, compared to those treated at pH 5.0. Assays of apoptotic cell death, including morphology, soluble DNA, caspase-3 activity and TUNEL also showed that as pH was increased, much greater HD concentrations were required to cause cell death. The modest decline in HD half-life measured in buffers of increasing pH, did not account for the protective effects of basic pH. The early event(s) that HD initiates to eventually culminate in cell death are not known. However, based on the data obtained in this study, we propose that HD causes an extracellular acidification through chemical hydrolysis and that this, in both a concentration and temporally related fashion, results in cytosolic acidification. Furthermore, HD also acts to poison the antiporter systems responsible for maintaining physiological pHi, so that the cells are unable to recover from this insult. It is this irreversible decline in pHi that initiates the cascade of events that results in HD-induced cell death.