Fluorescence microscopy has been used to study the cell surface distribution of the complement receptor for C3bi (CR3) on human neutrophils during locomotion. CR3 is an integral membrane protein that participates in cell attachment phenomena including chemotaxis. Fluorescein-and rhodamine-conjugated monoclonal IgG or Fab fragments were used to label CR3. We have previously shown that CR3 is uniformly distributed on unstimulated cells. During cell locomotion the fluorescent labels redistribute to the uropod and retraction fibers. To better understand the role of CR3 in chemotaxis, we have performed sequential two-color labeling experiments in conjunction with fluorescence microscopy. Double-labeling experiments were conducted by labeling adherent neutrophils with fluorescein-conjugated anti-CR3 followed by chemotaxis in a gradient of FMLP (lo-' M). The cells were then labeled again with rhodamine-conjugated anti-CR3. The uropod and distal training filopodia were labeled with fluorescein, whereas the cell body and occasionally proximal filopodia near the uropod were labeled with rhodamine. When neutrophils were fixed and permeabilized prior to the second CR3 labeling, the second fluorescent label was localized to a granulelike compartment(s), often near the lamellipodium. The results suggest a flow of CR3 from intracellular granules + lamellipodia and cell body + uropod + trailing filopodia during chemotaxis.