Adaptation of amoebae to four cooling tower biocides, which included a thiocarbamate compound, tributyltin neodecanoate mixed with quaternary ammonium compounds (TBT/QAC), another QAC alone, and an isothiazolin derivative, was studied. Previously we found that amoebae isolated from waters of cooling towers were more resistant to cooling tower biocides than amoebae from other habitats. Acanthamoeba hatchetti and Cochliopodium bilimbosum, obtained from American Type Culture Collection and used in the previous studies, were tested to determine whether they could adapt to cooling tower biocides. A. hatchetti was preexposed to subinhibitory concentrations of the four biocides for 72h, after which they were tested for their resistance to the same and other biocides. C. bilimbosum was exposed to only two biocides, as exposure to the other two was lethal after 72 h. Preexposure to the subinhibitory concentrations of the biocides increased the resistance of the amoebae, as indicated by a significant increase in the minimum inhibitory concentration (up to 30-fold). In addition, cross-resistance was also observed, i.e., exposure to one biocide caused resistance to other biocides. These results show that amoebae can adapt to biocides in a short time. The phenomenon of cross-resistance indicates that regularly alternating biocides, as is done to control microbial growth in cooling towers, may not be effective in keeping amoeba populations in check. On the contrary, exposure to one biocide may boost the amoebae's resistance to a second biocide before the second biocide is used in the cooling tower. Since amoebae may harbor Legionella, or alone cause human diseases, these results may be important in designing effective strategies for controlling pathogens in cooling towers.