Cytosolic free Ca2+ concentration was measured and imaged in human neutrophils moving towards a source of formylated peptide in a micropipette held close to the cells. Under these conditions, neutrophils changed shape and displayed chemotaxis without significant or persistent global or localised elevations in cytosolic free Ca2+. A rear-to-front persistent Ca2+ gradient of less than 0.5 nM/micron was present in the migrating neutrophils, until they reached the zone of higher peptide concentration, when an abrupt rise in cytosolic free Ca2+ concentration was triggered and chemotaxis stopped. Small localised rises in cytosolic free Ca2+, which were occasionally observed during neutrophil manoeuvring, were attributed to the effect of local deformation of the neutrophil membrane, since deformation of the membrane with a blunt micropipette caused similar Ca2+ changes. These data suggest that neutrophil chemotaxis towards a source of formylated peptide occurs without significant changes in Ca2+ signalling.