Neutrophils are capable of undergoing rapid directed movement up a concentration gradient of chemoattractant culminating in the phagocytosis of a target. We have developed a system to make rapid photometric measurements and ratio images of cytosolic free calcium [( Ca2+]i) in human neutrophils loaded with the fluorescent Ca2(+)-sensitive indicator Fura-2 during these processes. In our system neutrophils undergo chemotaxis toward and phagocytosis of IgG and IgM-coated sheep erythrocytes attached to a surface. During chemotaxis and phagocytosis, repetitive transients in [Ca2+]i take place. Accompanying the transients during phagocytosis is a localized [Ca2+]i increase in the periphagosomal region. This localized increase is more apparent in cells phagocytosing particles coated with both IgG and IgM than with IgM alone. No consistent localization of increased [Ca2+]i is seen in cells solely undergoing chemotaxis. The imaging techniques described here allow the observation of [Ca2+]i changes over regions of several microns 2 in a cell with a time resolution of approximately 0.5 s. [Ca2+]i gradients extending over regions greater than approximately 4 microns 2 and lasting at least 1 s can be reliably detected.