Technical Abstract: Spores of Encephalitozoon hellem, Encephalitozoon cuniculi, and Encephalitozoon intestinalis were heated to temperatures up to boiling or exposed to chlorine or70% ethanol and others were frozen. After each treatment, spores were inoculated into cell cultures to test for infectivity. After exposure to 0.54, 2.55, and 25.7 mg/L chlorine for 10 min, E. hellem, E. cuniculi, and E. intestinalis, respectively, were rendered noninfectious. Ethanol rendered E. cuniculi and E. intestinalis noninfectious after exposure for 5 min and E. hellem for 10 min. After freezing in water at -20 C for 8 h all 3 species were rendered noninfectious. Inhibition of E. cuniculi, E. intestinalis and E. hellem required exposure for 2, 5, and 10 min, respectively, at 100 C. These data indicate that ethanol is effective for surface disinfection of microsporidia and chlorine is useful for water disinfection. Freezing and heating in water reduce microsporidia infectivity but are time and temperature related.