The relationship of N-formyl-methionyl-leucyl-phenylalanine-stimulated Na+/H+ exchange to the chemotactic responsiveness of human neutrophils was investigated. The pHi changes, measured from the equilibrium distribution of 5,5-dimethyloxazolidine-2,4-dione, were correlated with the migratory behavior of the cells as assessed by the leading front method. Exposure of cells to 10 nM FMLP caused activation of Na+/H+ exchange, leading to a rise in pHi from approximately 7.25 to approximately 7.75. This intracellular alkalinization was inhibited by amiloride and by three more potent analogues. All four compounds reduced the chemotactic response to FMLP with apparent Ki values similar to those for inhibition of the pHi transients, thereby suggesting that the blocking effect of the drugs on directed cell migration was related to inhibition of Na+/H+ exchange. The effect was specific for stimulated cell locomotion: FMLP-induced chemotaxis and chemokinesis were inhibited in parallel, whereas random motility was unimpaired. The relationship of pHi to function was also studied as the pHi of FMLP-activated cells was varied between 6.8 and 8.6 by altering the chemical gradients for Na+ and H+ across the cell membrane. There was a direct, positive correlation between the pHi value attained following FMLP-stimulation and the locomotor response to a chemotactic gradient. These results indicate that the motile functions of human neutrophils can be regulated by their pHi.