Dictyostelium discoideum amoebae possess eight different actin crosslinking proteins. Immunofluorescence microscopy has been employed in this study to investigate the intracellular localization of two of these proteins, alpha-actinin and the 30 kD actin-bundling protein, to investigate whether they are redundant, or alternatively, make distinct contributions to cell structure and movement. The 30 kD protein is concentrated in the cleavage furrow of dividing cells, while enhanced staining for alpha-actinin is not apparent in this region. By contrast, alpha-actinin is concentrated around the contractile vacuole, while the 30 kD protein is not preferentially localized in the area of this organelle. Association of alpha-actinin with the contractile vacuole was confirmed by colocalization with calmodulin, a marker of this organelle. There are temporal differences in the localization of the 30 kD protein and alpha-actinin during phagocytosis. The 30 kD protein is localized in the phagocytic cup, but disassociates from phagosomes soon after internalization [Furukawa et al., 1992: Protoplasma 169: 18-27]. alpha-actinin enters the phagocytic cup after the 30 kD protein, and remains associated with the phagosome after the 30 kD protein has disassociated. These results support the hypothesis that alpha-actinin and the 30 kD protein play distinct roles in cell structure and movement in Dictyostelium.