Phagocytosis of opsonized particles by neutrophils involves highly localized alterations in the actin cytoskeleton that result in the formation of prominent pseudopodia and the phagocytic cup. Immunofluorescence microscopy was employed to monitor the distribution of several proteins that can regulate the cytoskeleton in human neutrophils undergoing phagocytosis of opsonized Candida albicans. The small GTPase Cdc42, its inhibitory subunit Rho-GDI, the adapter protein Nck, gamma-p21-activated protein kinase (gamma-Pak), and cofilin were found to undergo rapid association with the developing phagosomes in these cells. In contrast, these signaling proteins were either diffusely distributed in the cytoplasm or enriched in focal points at the base of the cell when optical sections were obtained from regions of the cell not involved in phagocytosis. These results are consistent with Cdc42 being critically involved in initiating the early events in phagocytosis by its ability to activate Pak and the Wiskott-Aldrich Syndrome protein that triggers the localized polymerization of actin. These data provide insights into the molecular mechanisms that trigger changes in the actin cytoskeleton during phagocytosis.