To describe the physiological impact of the Na+ /H+ exchanger NHE1 on cell migration we examined distribution pattern and activity of NHE1 in human melanoma cells. An NHE1 deficient mutant was generated from wild type cells employing an H+ - suicide technique. This NHE1 deficient cell line was retransfected with NHE1 and then called the "rescued" clone. NHE1 could be localized at the leading edge of migrating wild type and rescued cells whereas it was not detectable in the NHE1 deficient clone. NHE1 activity of wild type, NHE1 deficient and rescued cells was therefore checked by determining the Na+ -dependent recovery of the intracellular pH (pHi) upon cytosolic acidification. The recovery rate was nearly the same in wild type and rescued cells (~0.2 pH units min-1 ). It was reduced by more than 50% in NHE1 deficient cells. Using the fluorescein conjugate DHPE for pH measurements at the outer leaflet of the cell membrane we found that wild type and rescued cells established an H+ gradient in the direction of movement. The H+ concentration was higher at the leading edge than at the rear end. This longitudinal gradient was not present in the NHE1 deficient mutant. As wild type and rescued cells migrate whereas the NHE1 deficient clone does not we conclude that NHE1 activity contributes to the generation of a longitudinal pH gradient needed for migration.