Members of the genus Acanthamoeba are present in diverse environments, from freshwater to soil, and also in humans, causing serious brain and corneal infections. Their life cycle presents two stages: the dividing trophozoite and the quiescent cyst. The structures of these life stages have been studied for many years, and structural data have been used for taxonomy. The ultrastructural work on Acanthamoeba cysts was carried out previously by routine transmission electron microscopy (TEM), a process that requires the use of chemical fixation, a procedure that can cause serious artifacts in the ultrastructure of the studied material. In order to prevent fixation artifacts, we processed Acanthamoeba polyphaga cysts by ultrarapid freezing, followed by freeze-fracturing and deep-etching, in order to obtain a 3D visualization of the arrangements of the cyst wall. The exocyst presented an irregular surface, with vesicles located within or near this layer. The endocyst, instead, showed a biphasic arrangement with a more compact district in its innermost part, and a more loosened outer layer. For this reason, it was difficult to distinguish the filaments present in the intercyst space from those forming the endocyst. Surprisingly, the intercyst space was thinner when compared with samples processed by conventional TEM, evidencing the possible damage consequent to the use of chemical fixation.