Human peripheral blood neutrophils obtained from healthy adults were examined in vitro. We assessed the effects of sequential stepwise increases in the concentration of the chemotactic dipeptide N-formyl-l-methionyl-l-phenylalanine (f-Met-Phe) on neutrophil attachment to serum-coated glass, detachment from serum-coated glass and the distribution on the cell surface of binding sites for albumin-coated latex beads. The initial exposure to f-Met-Phe resulted in increased adhesiveness and binding of latex beads in a random pattern over the cell surface. The second exposure to f-Met-Phe resulted in decreased adherence, detachment of neutrophils from serum-coated glass, and movement of binding sites for latex beads to the uropod. Enhanced adhesiveness and redistribution of binding sites were blocked by 0.1 mM N-α-p-tosyl-l-lysine chloromethyl ketone, a concentration that did not reduce the change in cellular shape caused by f-Met-Phe. Cytochalasin B (5 μg/ml) blocked the redistribution of binding sites as well as the change in shape. The third exposure to f-Met-Phe was given along with the latex beads. The stimulus was stopped after 2 min by fixing cells in suspension with glutaraldehyde. If the third exposure was at a concentration higher than the second, the beads were bound in the region of the lamellipodia in 70% of the cells. If lower, binding to the lamellipodia was found in a significantly smaller proportion of cells (13%). The results support the concept that neutrophils develop a polarized distribution of f-Met-Phe-induced adhesion sites in response to increasing concentrations of f-Met-Phe, and these sites flow from the region of the lamellipodia to the uropod.