TheDictyostelium discoideum 30 kDa actin-bundling protein cross-links actin filaments into bundles in vitro, and is present in filopodia and pseudopodia in living cells. Monoclonal antibodies reactive with this protein have been isolated, and employed as specific probes for the function of this protein. The monoclonal antibody B2C blocks the interaction of the 30 kDa protein with F-actin in vitro, and decreases phagocytosis ofE. coli when introduced into livingDictyostelium cells by controlled sonication. Use of this monoclonal antibody for visualization of the 30 kDa protein by immunofluorescence microscopy reveals striking localization around food particles during the process of phagocytosis. Double staining with rhodamine-labelled phalloidin and the monoclonal antibody documents the co-localization of the 30 kDa protein and actin during formation of phagocytic cups. The dissociation of the 30 kDa protein occurs during the process of maturation to form phagolysosomes. These results support the hypothesis that this actin cross-linking protein participates in dynamic rearrangements of actin filaments accompanying phagocytosis.