Melanoma cells in vivo maintain intracellular pH (pH_i) in a viable range despite an extracellular tumor pH (pH_e) that is typically below 7.0. In general, three families of transporters are capable of removing metabolic protons, but the specific transporters responsible for the maintenance of pH_i at low pH_e in melanomas have not been identified. Although the transporters exist in most cells, an inhibitor would be predicted to have selectivity for cells located in an acidic tumor bed because cells in that environment would be expected to have transporters chronically activated. In this report, the levels and extent of expression of the Na⁺/H⁺ exchanger (NHE-1) and two of the H⁺-linked monocarboxylate transporters (MCTs) were evaluated in three melanoma cell lines. The effects of inhibitors of each transporter were tested at an extracellular pH (pH_e) of 7.3, 6.7, or 6.5 in melanoma cells that were grown at pH_e 7.3 or 6.7. The activity of MCT isoform 1 (MCT-1) was up-regulated in three melanoma cell lines at low pH_e, but that of NHE-1 was not. Furthermore, NHE-1 activity was lower in the melanomas than in other normal and malignant cell lines that were tested. Reverse transcription-PCR using primers specific for MCT-1, MCT-4, and NHE-1 showed that expression of none of these transporters was reproducibly up-regulated at the level of transcription when cells were grown at pH_e 6.7 instead of pH_e 7.3. Ex vivo experiments using DB-1 human melanoma xenografts grown in severe combined immunodeficient mice found that MCT-1 and not NHE-1 was a major determinant of DB-1 tumor cell pH_i. Taken together, the data indicate that MCTs are major determinants of pH regulation in melanoma. In contrast, keratinocytes and melanocytes under low pH_e conditions relied on NHE-1. Inhibitors of MCTs thus have great potential to improve the effectiveness of chemotherapeutic drugs that work best at low pH_i, such as alkylating agents and platinum-containing compounds, and they should be selective for cells in an acidic tumor bed. In most tissues, it is proposed that the NHE-1 could compensate for an inhibited MCT to prevent acidification, but in melanoma cells this did not occur. Therefore, MCT inhibitors may be particularly effective against malignant melanoma.