AbstractDirected movement of polymorphonuclear neutrophils (PMN) requires cell polarization and the orderly making and breaking of cell‐substrate contacts. We compared the movement of human PMN suspended from the underside of glass coverslips to that of PMN seen in “profile” on fibers, using brightfield, differential interference contrast and reflection interference microscopy. Images were recorded on film and videotape and analyzed in real time and time lapse. The distribution of F‐actin was observed with image‐enhanced fluorescence microscopy after staining with NBD‐phallacidin.PMN exhibited two patterns of motility. Fifteen to twenty‐five percent of cells moved in a low profile gliding pattern and exhibited cauded displacement of dorsal surface folds. Most PMN made progress by cycles of partial release of the lamellipodium from the substrate and anterior advance followed by arching or rolling and lamellipodial reassociation with the substrate. Cells stimulated with bacteria, casein, or chemotactic formyl peptide rarely spread on the coverglass but waved into the medium attached only by the uropod. Eventually, many detached completely from the substrate. Cells confined to the substrate surface with overlying agarose were able to locomote when confronted with these substances.F‐actin was irregularly distributed in nonpolarized suspended cells but concentrated in the lamellipodium in polarized cells. As cells arched along a substrate, F‐actin accumulated in foci corresponding to the substrate‐PMN interface, particularly at the uropod and retraction fibrils. Conversely, cells that were physically restricted to movement in the plane of the substrate surface by overlying agarose exhibited diffuse F‐actin along the entire cell. Suspended PMN polarized with formyl peptide and incubated with Con A accumulated F‐actin at the uropod. These observations suggest that both PMN locomotion and the movement of Con A binding sites involve the caudad re