Characterization of early Ca2+ signalling events is crucial for understanding the mechanisms which lead to cell signalling. Rapid confocal laser scanning of Fluo3-loaded neutrophils was used to provide spatially resolved cytosolic free Ca2+ measurements from neutrophils stimulated with formyl-Met-Leu-Phe with a time resolution of 12.5 ms. Heterogeneity of the magnitude and timing of the response was observed between individual neutrophils. There was always a delay between contact with the stimulus and onset of the Ca2+ signal, with a minimum delay of 75 ms and a mean delay of 530 ms (n = 150). There was no delay in the cytosolic free Ca2+ rise induced by ionomycin. The earliest Ca2+ event detected following stimulation with formyl-Met-Leu-Phe (1 microM) was a localized rise in cytosolic free Ca2+ from a location within the cell, pointing to Ca2+ store release as the initial event for triggering whole-cell Ca2+ changes in these cells.