Repeated transient increases in intracellular free calcium levels ([Ca2-]i) are required for polymorphonuclear neutrophil migration on two-dimensional surfaces coated with fibronectin or vitronectin. Cells in which [Ca2+]i is buffered with quin2 become stuck on these substrates. Neutrophils migrating through the extracellular matrix in vivo encounter these and other substrates in a three-dimensional architecture that may alter the spatial distribution of adhesion receptors in contact with the matrix. In this study, we used fluorescence confocal microscopy to obtain moving three-dimensional images of neutrophils migrating through a biological tissue (human amnion) in the presence and absence of [Ca2+]i-buffering with quin2. In the absence of buffering, [Ca2+]i transients similar to those seen in cells migrating in two-dimensions were observed. [Ca2+]i-buffered neutrophils were able to migrate into the matrix, but they became attached firmly to the substrate at the rear of the cell, resulting in a drastically elongated morphology. Immunofluorescence revealed that neutrophils adhered to regions of the matrix that contained fibronectin. RGD-containing peptides and antibodies that block integrin adhesion receptors for fibronectin and vitronectin were able to rescue the migration of quin2-treated cells through three-dimensional gels containing fibronectin and vitronectin. These data show that neutrophils migrating through physiologically relevant, three-dimensional matrices undergo repetitive increases in [Ca2+]i that are required for integrin-mediated detachment from the matrix.