Background: The Na+/H+ exchanger isoform 1 (NHE1) is believed to be essential for directional cell migration during wound healing and tumour invasiveness. However, in the intestinal epithelium, which displays very rapid cellular restitution of surface wounds, other NHE isoforms (NHE2, NHE3) are expressed far more strongly. Aim and methods: We studied the role of NHE isoforms 1, 2 and 3 on wound healing velocity in RGM1 cells, a nontransformed rat gastric surface cell line and in Caco2bbe cells, a well differentiating human colonic tumour cell line. Migration velocity was studied during growth factor-stimulated and low pH-stimulated as well as serum starvation-inhibited wound healing. To assess cell migration speed objectively, a fluorescent dye-based plate reader assay was used [1]. NHE1 and 2 were dose-dependently inhibited by HOE642 [2], whereas NHE1 and 3 were selectively inhibited by S1611 [3]. 5-(N,N-Dimethyl)amiloride (DMA) was used to inhibit Na+/H+ exchangers less specifically [4]. Stable NHE3 overexpression was performed in Caco2bbe cells, while lentiviral transient NHE2 overexpression was achieved in RGM-1 cells. Results: In confluent monolayers, RGM1 cells express NHE1 mRNA in far higher levels than other NHEs, whereas NHE2 mRNA expression is higher than NHE1 and NHE3 in Caco2bbe cells. Both cell lines displayed cell migration after experimental wounding even after serum starvation, where migratory speed was independent of NHE activity in either cell line. Fetal calf serum (FCS) significantly stimulated cell migration in a partly NHE1-dependent fashion in RGM-1 cells, but in a NHE-independent fashion in Caco2bbe cells. Preincubation at low pH also stimulated migratory speed in a NHE1-dependent fashion in both RGM-1 and Caco2bbe cells. NHE2 overexpressing RGM-1 cells migrated more slowly in the presence of FCS, and more so after low pH preincubation, which was reversed by pharmacological NHE2 inhibition. NHE3 overexpression as well as inhibition did not result in a change in migratory speed. DMA impaired cell migration more strongly than Na+/H+-exchange inhibition could account for, implying nonspecific effects of the amiloride analogue. Conclusions: Wound closure is possible even in the absence of NHE functional activity in gastrointestinal epithelial cells. Dependency of cell migration velocity on NHE1 varies among cell types. Low pH enhances cell migration in a NHE1-dependent fashion, whereas stimulation by growth factors is predominantly NHE1-independent. NHE2 slows cell migration speed. Therapeutic NHE inhibition for gastrointestinal malignancy is therefore likely to have a highly variable effect on tumour invasiveness depending on the cell type.