Recently, it has been suggested that the cellular uptake of chemotherapeutic drugs may be dependent on the pH gradient between the intracellular (pHi) and extracellular (pHe) compartments. It has been demonstrated in murine tumor models that the extracellular environment is acidic, relative to the intracellular environment, thus favoring preferential accumulation of drugs that are weak acids into cells. However, concomitant measurements of pHi and pHe in spontaneous tumors have not been reported, so it is not certain how well the murine results translate to the clinical scenario. In this study, both types of measurements were performed in dogs with spontaneous malignant soft tissue tumors. On average, pHe was more acidic than pHi, with maintenance of a more physiologically balanced intracellular tumor environment. However, the magnitude of the gradient varied widely, and individual tumors had both positive and negative pH gradients (pHi - pHe). These data suggest that the magnitude and direction of the pH gradient may need to be measured for individual patient tumors and/or that manipulation of pHe may be required if exploitation of the pH gradient is to be achieved for tumor-selective augmentation of intracellular drug delivery.