SUMMARYThe factors that characterizeAcanthamoebastrains as harmless or potentially pathogenic have not been elucidated. Analysing thein vitroandin vivoparameters ofAcanthamoebasamples, including heat tolerance at temperatures close to that of the human body, cytopathic effects, and their ability to cause infections in animals, has been proposed to identify their pathogenic potential. Another promising criterion for differentiating strains is the analysis of their biochemical and immunochemical properties. In this study, a comparative evaluation between clinical and environmentalAcanthamoebaisolates was performed on the basis of physiological, morphological, and immunochemical criteria. Crude antigens were used to characterize the protein profiles by electrophoresis and immunize mice to produce polyclonal and monoclonal antibodies. The antibodies were characterized using ELISA, Western blotting, and immunofluorescence techniques. The results obtained with polyclonal antibodies suggest the presence of specific proteins for each studied isolate and co-reactive immunochemical profiles among conserved components. Ten monoclonal antibody clones were obtained; mAb3 recognized 3 out of 4 samples studied. The results of this study may help standardize criteria for identifying and characterizingAcanthamoebastrains. Taken together, our results support the view that a set of features may help differentiateAcanthamoebaspecies and isolates.