Pathophysiological factors exist within solid tumors that lead to a low pH environment. Therefore, pH-sensitive cancer chemotherapeutic agents may selectively target and kill tumor cells while sparing the normal tissue from toxicity. Using colony forming assays to assess cell survival, we found that EMT6 mouse mammary tumor cells were more sensitive to cisplatin cytotoxicity when cultured in pH 6.0 medium than in pH 7.2, 6.8, or 6.4 medium. The pH-dependent cytotoxicity of cisplatin resulted from an increase in cisplatin accumulation and an increase in the amount of DNA cross-links at pH 6.0 compared with pH 7.2. Because DNA is the cytotoxic target of cisplatin, intracellular pH (pHi) may be an important factor in determining the cytotoxicity of anticancer drugs at low extracellular pH (pHe). Therefore, manipulating the pHi of cells could be one method to enhance the effectiveness of the pH-sensitive chemotherapeutic agents. The pHi of EMT6 cells varied with pHe: at pHe 7.2, pHi was 7.54; at pHe 6.8, pHi was 7.29; at pHe 6.4, pHi was 7.02; and at pHe 6.0, pHi was 6.64. Using inhibitors to the ion transport mechanisms which regulate pHi, 5-N,N-hexamethylene amiloride (NHMA) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), the pHi and pHe of EMT6 cells were equilibrated. To evaluate the importance of pHi in determining drug toxicity, cell survival was determined for cells treated with cisplatin in the presence of NHMA and SITS. Cells cultured with NHMA and SITS were less sensitive to cisplatin. The cisplatin resistance obtained was independent of pH and could be attributed to the presence of SITS.