The objective of this study was to evaluate the fate of infectious prions in water. Prion diseases such as bovine spongiform encephalopathy also known as "mad cow disease", can be spread by ingestion of animal tissue and feces, suggesting that transmission via water may be possible. Few studies have evaluated the fate of prions in water and most have used assays that were not capable of determining whether or not the prions were infectious. In this study, known concentrations of infectious prions (PrP Sc) were added to deionized water, tap water, and treated wastewater. Water samples were incubated at mesophilic and thermophilic temperatures for up to for 8 weeks. The standard scrapie cell assay (SSCA) and the ELISPOT (Enzyme Linked Immuno-Spot) reaction was performed to determine the quantity of infectious prions that survived incubation. A large reduction of PrP Sc was observed during the course of the experiments at every temperature. A maximum rate of inactivation in water occurred under thermophilic conditions. The results suggest that temperature and dissolved organic matter influence inactivation. Previous studies report that prions were very resistant to degradation; however, the methods used did not distinguish whether or not prions were infectious. Our data show that infectious prions are susceptible to inactivation in water.