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Purpose: The aim of this study was to assess the presence of pathogenic prions in the brain tissue of eye donors and to evaluate the benefits of 2-year obligatory testing in the Czech Republic., Methods: Brain tissue was retrieved during autopsies of eye donors of 3 tissue banks in the Czech Republic. The frozen specimens obtained from the frontal lobe were transported to the Czech National Reference Laboratory for the diagnosis of human prion disorders. The presence of pathogenic prions was tested using the Prionics-Check WESTERN kit. Confirmative Western blotting using 1 of 2 different clones of monoclonal anti-PrP antibody was performed as well., Results: No pathogenic prions were found in any of the 1142 tested specimens. One specimen revealed weak positivity at initial screening; however, repeated examination of the specimen and other specimens from different locations in the brain of the same donor did not confirm the presence of pathogenic prions. The negative result was confirmed by the National CJD Surveillance Unit, University of Edinburgh, United Kingdom., Conclusion: The absence of pathogenic prions from all of the 1142 tested specimens corresponds to the presumed very low risk of transmission of Creutzfeldt-Jakob disease through corneal graft transplantation. As a result of this disorder's rarity, a larger series of tested samples should be evaluated to obtain statistically significant findings. Although such testing increases the safety of donor eye tissue, it also increases the expense, causes organizational difficulties, and may extend the time needed to release the tissue for grafting.