The effects of extracellular pH (6.2 to 7.3) on uptake and cytotoxicity of the weak acid anti-tumor drug chlorambucil were investigated. Decreasing extracellular pH from 7.3 to 6.5 had a negligible effect on the intracellular pH of Chinese hamster V79 fibroblasts, thus resulting in the formation of a transmembrane pH gradient (intracellular alkaline). Addition of high concentrations of acetate or bicarbonate partially collapsed the pH gradient. Chlorambucil (pKa = 5.8) behaved as a weak acid with enhanced accumulation and cytotoxicity at extracellular pH less than 7.0. As predicted for a weak acid, partial collapse of the transmembrane pH gradient decreased both uptake and cell killing. Since the interstitial pH of micrometastases and solid tumors of many cancers is low relative to normal tissues, these results have potential implications for both in vitro drug testing and in vivo therapy.