Acanthamoeba species infect humans occasionally and act as opportunistic pathogens in immunocompromised individuals. This study demonstrates the application of cytocentrifugation as an aid to identification of Acanthamoebae. In addition, certain staining procedures clearly optimized visualization of characteristic amoebic features. This was demonstrated by adding amoebae from laboratory cultures to bronchoalveolar lavage specimens. In preparations stained by the Papanicolaou, trichrome, or hematoxylin-eosin (H&E) procedures, the discrete deeper staining nucleolus was the most distinctive feature. The vacuolated cytoplasm also aided in the identification of amoebae. These features were less apparent and often distorted following staining of Acanthamoeba species with the Hema III and Giemsa procedures.