Fluorescence confocal microscopy was used to obtain three-dimensional (3-D) images of human neutrophils migrating through a 3-D matrix of amniotic membrane with a temporal resolution of 30-60 s and a spatial resolution of approximately 2 microm in the z-dimension. Neutrophils migrating in response to a chemoattractant gradient within a 3-D matrix were apparently able to generate traction by use of lateral pseudopods inserted into footholds in the matrix as evidenced by matrix distortion. Similar anchored pseudopods were seen in cells migrating across polycarbonate membranes with 0.8-microm pores; the presence of these pores increased cell polarization and migration compared with cells on membranes without pores. Expansion of pseudopods distal to narrow constrictions in the matrix and porous filters was observed and appeared to be used to pull cells through the openings. Neutrophils deformed parts of the elastic amnion matrix during migration without permanently altering the substrate. Contact guidance of neutrophils crawling along matrix fibrils was also observed. These observations show that neutrophils migrating in 3-D are able to utilize mechanical structures in the matrix, not present on 2-D surfaces, to generate traction for locomotion.