Neutrophils expend large amounts of energy to perform demanding cell functions. To better understand energy production and flow during cell activation, immunofluorescence microscopy was employed to determine the location of the key metabolic enzyme hexokinase during various conditions. Hexokinase is translocated from the neutrophil's cytosol to its periphery in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and other activating stimuli, but not during exposure to the formyl peptide receptor antagonist N-tert-BOC-phe-leu-phe-leu-phe (Boc-PLPLP). Translocation was observed from 10(-6) to 10(-9)M fMLP. However, fMLP did not affect the intracellular distribution of lactate dehydrogenase. Hexokinase accumulated at the lamellipodium of cells exposured to an fMLP gradient whereas it localized to the phagosome after latex bead uptake. Thus, hexokinase is differentially translocated within cells depending upon the prevailing physiological conditions. Further studies noted that cytochalasin D, dexamethasone, and indomethacin blocked hexokinase translocation. Parallel regulation of reactive oxygen metabolite (ROM) production was shown. We speculate that hexokinase translocation participates in neutrophil activation.