It has been reported that the virulence of axenically cultivated Entamoeba histolytica increases following growth with cholesterol. The purpose of this study was to determine whether cholesterol would enhance the virulence of axenically cultivated Naegleria fowleri. Amoebae were cultivated in axenic medium with (100 pg/ml) or without cholesterol for 6 months and tested in mice for changes in virulence. After 6 months of continuous cultivation, N. fowleri grown with cholesterol was less virulent for mice than the same strain grown without cholesterol.