Three actin-bundling proteins have been isolated from Dictyostelium discoideum cells. To investigate whether these proteins might play different roles in Dictyostelium cells, the distribution of these proteins was examined in cells of the slime mold at various developmental stages by agar-overlay immunofluorescence. ABP-30a was localized in pseudopods and projections, and regions of intercellular contacts, where filaments of actin were present at high levels. ABP-30a was not concentrated in the cleavage furrow of dividing cells or in the tail cortical regions of actively moving cells even though actin was present at high levels in both regions. Some ABP-50 was concentrated at the leading edges of pseudopods and at the distal ends of projections, but most ABP-50 was distributed diffusely in the cytoplasm. ABP-30b was not concentrated in any restricted regions and was diffusely distributed in the cytoplasm. When developed cells were stimulated with cAMP, fluorescence due to ABP-30a and ABP-50 increased in the cortical region 5 to 10 sec after stimulation. Quantitative analysis of levels of both proteins in Triton-insoluble cytoskeletons after stimulation by cAMP also showed that levels of both proteins increased within 5 to 10 sec. These observations indicate that levels of ABP-30a and AMP-50 are regulated by signal transduction during chemotaxis and that the three actin-bundling proteins play different roles in Dictyostelium cells.