Previous studies showed that the activity of the Na+ /H+ exchanger NHE1 is required for human melanoma cell migration. Our long-term objective is to repress tumor cell migration and thus metastasis by NHE1 inhibition in vivo (mice). We therefore investigated in vitro what effects extracellular pH (pHe) and NHE1 activity have on mouse melanoma (B16) cell adhesion, migration and invasion. Expression of NHE1 in B16 cells was shown by Western blotting. NHE1 activity was demonstrated by measuring the intracellular pH (pHi) prior to and upon NHE1 inhibition with the specific NHE1 inhibitor cariporide (HOE642). Addition of HOE642 led to a reversible decrease in pHi. Adhesion of B16 cells on a matrix consisting of collagen I, collagen IV and laminin (basal membrane-like matrix) was significantly reduced by blocking the NHE1 with HOE642. Without NHE1 inhibition maximum adhesion occurred between pHe 6.4 and 6.8. Cells were most motile and invasive at pHe 7.0. These results show that NHE1 activity plays the same role in mouse melanoma cells (B16) as it does in human melanoma cells. The use of B16 cells for in vivo studies is therefore justified.