To study the spatial and temporal regulation of the actin cytoskeleton, we have analyzed the actin concentration dynamics in live Dictyostelium. The relative actin concentration was analyzed with respect to cell behavior by fluorescence morphometry. We electroporated rhodamineactin into Dictyostelium cells and acquired images with 200–300 millisecond temporal and ∼250 nm spatial resolutions. To convert fluorescence intensity into actin concentration, the observation was made on nearly two-dimensional cells, and the actin signal was ratioed over a volume marker (FITC-BSA or GFP). Since the emission of FITC and GFP is pH-dependent, we first measured the cytoplasmic pH in live cells and determined that the pHi in pseudopods is same as that of general cytoplasm. During cytokinesis, the relative concentration of actin in the cleavage furrow was significantly higher than in the general cytoplasm. In migrating cells, actin was recruited surprisingly rapidly, particularly in the pseudopod. We found that the region of high actin concentration moves relative to the leading edge when a pseudopod projects or retracts. When the pseudopod retracts, the actin density dissipates within 5 seconds. We have also found that actin accumulates in developing pseudopods in an oscillatory manner, and this timing coordinates with advancement of the centroid. This is the first study to reveal the dynamic changes in relative concentration of actin in live cells and to quantitatively correlate these changes with the locomotive behavior of the amoeba.