Actin from Dictyostelium was labeled with iodoacetamide tetramethylrhodamine (IAR). The labeled actin retained the ability to polymerize into filaments. The labeled actin was introduced into Dictyostelium cells by electroporation. The introduced IAR-labeled actin was diffusely distributed in the cytoplasm but some of it was concentrated in small and large projections at the cell periphery. IAR-labeled actin was concentrated in pseudopods and in the tail cortical region in actively migrating cells. Intense fluorescence due to labeled actin appeared rapidly and disappeared during the extension and retention of pseudopods. During cytokinesis, IAR-labeled actin was concentrated in both polar regions and slightly concentrated in the furrow region. However, the ratio of intensities due to IAR-labeled actin and fluorescently labeled bovine serum albumin that had been introduced simultaneously into cells showed the absence of any concentration of IAR-labeled actin in the furrow region. Staining of fixed cells with fluorescently labeled phalloidin revealed that filamentous actin was rich in the furrow region. These observations indicate that actin is concentrated at this region in higher ratio of filamentous actin to monomeric actin than in other regions of the cytoplasm. Image analysis revealed that the concentration of IAR-labeled actin was high in a pseudopod of an actively migrating cell. Comparisons with staining by fluorescently labeled phalloidin of fixed cells revealed that there was a decreasing gradient in the concentration of filamentous actin from the tip to the base of a pseudopod. These results reveal the high rate at which actin is coordinately organized during mitosis and locomotion in highly motile Dictyostelium cells.