The intracellular localization of pyridoxal phosphatase activity was demonstrated in human neutrophils by electron microscope cytochemistry. Under alkaline conditions, an enzyme active against pyridoxal phosphate was localized to a cytoplasmic granule population, the phosphasome. These granules have previously been shown by electron microscope cytochemical techniques and by subcellular fractionation to be rich in alkaline phosphatase. Under acidic conditions, a phosphatase activity against pyridoxal phosphate was localized to intracellular multilamellar bodies resembling secondary lysosomes. These were quite distinct from the primary, secondary and phosphasome granules and this unique localization corresponds to that previously demonstrated (tertiary granules) by subcellular fractionation studies of these cells. The similarity in the enzyme reaction requirements of alkaline pyridoxal phosphatase and alkaline phosphatase, and their localization to the same subcellular organelle, suggests that pyridoxal phosphate may be a physiological substrate for human neutrophil alkaline phosphatase.