Neutrophils are the first immune cells to arrive at a site of inflammation. Acidification of the local environment is one of the major stress factors which immune cells face while approaching the core of the inflamed tissue. It is now well known, that most neutrophil functions such as ROS production, phagocytosis and apoptosis are influenced by extracellular pH (pHe). Since activated neutrophils often have to cope with harsh pHe conditions, they display multiple regulatory mechanisms to maintain their functions. In our project, we study the role of the Na+/H+-exchanger (NHE1) in murine (C57BL/6 strain) neutrophil migration and intracellular pH homeostasis upon chemoattractant stimulation in an acidic environment. Neutrophil chemotaxis was monitored in a 3-dimensional collagen I matrix using Ibidi µ-Slides. Live-cell imaging was performed for 30 min and images were acquired in 5s intervals. Chemotaxis was induced in gradients of different concentrations of the complement component C5a in the absence or presence of the NHE1 inhibitor cariporide. pHe in the chemotaxis chambers ranged from pHe 6.5 to pHe 7.6. Intracellular pH (pHi) of neutrophils was measured using the ratiometric pH indicator BCECF. Cells were seeded onto a fibronectin coating and superfused with Ringer’s solution (pH 7.4 or pH 6.5) containing C5a without/with NHE1 inhibitor. All values are given as means ± SEM, compared by ANOVA. Speed and directionality of migrating neutrophils are decreased upon NHE1 inhibition (mean speed [µm/min] 11.8 ± 0.3 vs control 14.7 ± 0.2). However, the velocity in the direction of the C5a gradient is affected much stronger upon NHE1 inhibition: It is reduced by ~70%. In pHe 6.5, the mean speed is reduced by ~27%, whereas the velocity towards the C5a gradient is lowered by ~76% (compared to values in pHe 7.0). Upon stimulation, pHi rapidly decreases (ΔpHi -0.21 ± 0.015), before stabilizing after ~2min. Simultaneous inhibition of NHE1 augments the acidification (ΔpHi -0.51 ± 0.04). In pHe 6.5, no compensatory stabilization is detected, whereas inhibition of NHE1 again enhances the acidification upon stimulation. Our results suggest a chemotaxis optimum at pHe 7.0, while NHE1 inhibition and a more acidic pHe impair directed migration. Notably, acid pHe and NHE1 inhibition affect primarily the directionality of migration, while the velocity is only slightly decreased. pHi is similarly affected by an extracellular acidification and by NHE1 inhibition. We therefore propose that the acidification at a site of inflammation decreases speed and directionality of the migrating neutrophil by challenging the intracellular pH homeostasis. Possibly, these mechanisms imply an immunological control of neutrophil chemotaxis, helping the cells to approach the inflamed tissue, but causing a migration halt when the neutrophils arrive at the acidic core of the inflammation.