Melanoma cell migration requires the formation and release of cell-matrix contacts mediated by adhesion molecules such as integrins. The Na⁺/H⁺ exchanger NHE1 accumulates at the cell front where it co-localizes with adhesion molecules. NHE1 activity facilitates cell migration by generating an intracellular pH (pH_i) gradient along the axis of movement which is mirrored by a complementary extracellular pH (pH_e) gradient at the cell surface. pH_i is higher, pH_e is lower at the cell front than at the cell rear. As cell adhesion depends on pH_e and NHE1 activity, we hypothesize that NHE1 modulates pH_i and pH_e very locally in the immediate vicinity of focal adhesions. Total Internal Reflection Fluorescence (TIRF) microscopy enabled us to selectively visualize regions at the basal plasma membrane of human melanoma cells (MV3). Focal adhesions were labeled with DsRed2-paxillin. Cells were seeded on collagen I. pH_i was measured ratiometrically at both focal adhesions and DsRed2-free regions using the proton-sensitive, fluorescent dye BCECF. To determine whether possible differences in pH between adhesive and non-adhesive regions depend on NHE1 activity the specific NHE1 inhibitor HOE642 was applied. The intracellular proton concentration was lower at focal adhesions (4.9x10^-8 mol/l ? pH 7.31) compared to DsRed2 free regions (6.4x10^-8 mol/l ? pH 7.2). At focal adhesions NHE1 inhibition by HoE642 caused a significantly stronger increase in the intracellular proton concentration (3.1-fold vs. 1.9-fold). We conclude that NHE1 is predominantly active at focal adhesions. Here, NHE1 contributes to the generation of pH nanoenvironments probably needed to modulate the strength of cell-matrix contacts.