Tumor cells in vivo often exist in an ischemic microenvironment that would compromise the growth of normal cells. To minimize intracellular acidification under these conditions, these cells are thought to upregulate H ฯฉ transport mechanisms and/or slow the rate at which metabolic processes generate intracellular protons. Proton extrusion has been compared under identical conditions in two closely related human breast cell lines: nonmalignant but immortalized HMT-3522/S1 and malignant HMT-3522/T4 -2 cells derived from them. Only the latter were capable of tumor formation in host animals or long-term growth in a low-pH medium designed to mimic conditions in many solid tumors. However, detailed study of the dynamics of proton extrusion in the two cell lines revealed no significant differences. Thus, even though the ability to upregulate proton extrusion in a low pH environment (pH e ) may be important for cell survival in a tumor, this ability is not acquired along with the capacity to form solid tumors and is not unique to the transformed cell. This conclusion was based on fluorescence measurements of intracellular pH (pH i ) on cells that were plated on extracellular matrix, allowing them to remain adherent to proteins to which they had become attached 24 to 48 h earlier. Proton translocation under conditions of low pH e was observed by monitoring pH i after exposing cells to an acute acidification of the surrounding medium. Proton translocation at normal pH e was measured by monitoring the recovery after introduction of an intracellular proton load by treatment with ammonium chloride. Even in the presence of inhibitors of the three major mechanisms of proton translocation (sodium-proton antiport, bicarbonate transport, and proton-lactate symport) together with acidification of their medium, cells showed only about 0.4 units of reduction in pH i . This was attributed to a slowing of metabolic proton generation because the inhibitors were shown to be effective when the same cells were given an intracellular acidification.