A non-radioactive method was used to investigate the uptake by Acanthamoeba castellanii of chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB). Based on the Giles et al. (1974) hypothesis, the uptake of CHA by trophozoites appeared to be of the L3 pattern whereas that of cysts was C2. Unlike CHA, trophozoites took up PHMB with an L2 pattern at low concentrations followed by a C-type pattern at higher concentrations, the uptake by cysts was found to be of the C2 pattern with a plateau effect at high concentrations. A diphasic leakage effect was found in trophozoites whereas a relatively low peak of maximal leakage occurred from cysts treated with high biocide concentrations. The amount of pentose release depended on the formulation ingredients. No correlation between pentose leakage and trophozoicidal or cysticidal activity was found.