The structures of purified ameba F-actin and cytoplasmic filaments of Acanthamoeba castellanii have been studied by electron microscopy. In negatively stained preparations, purified ameba F-actin is a beaded filament about 60Åin diameter and up to 3 μ long, with a double helical substructure similar to muscle F-actin. When reacted with rabbit muscle heavy meromyosin, ameba F-actin forms a complex consisting of a periodic linear array of ‘arrowheads’ with a spacing of 370Åthat is indistinguishable from the complex between rabbit muscle F-actin and heavy meromyosin.The long thin 80Åfilaments that are seen in electron micrographs of thin sections of untreated Acanthamoeba are still present after extraction of the amebas with glycerol. After treatment of the glycerinated amebas with the heavy meromyosin, distinct arrowhead complexes form along the filaments. This reaction specifically identifies the filaments as actin, first, because it can be blocked by Mg-pyrophosphate or Mg-ATP, compounds which are known to dissociate actin and heavy meromyosin and second, because the reaction does not occur with the non-specific mixture of proteins found in goat serum. The identification of the cytoplasmic filaments as F-actin strongly suggests that the filaments participate in the movement of the ameba.