The survival of Encephalitozoon cuniculi Levaditi, Nicolau et Schoen, 1923 spores suspended in distilled water and exposed at defined temperatures was investigated. Infectivity of E. cuniculi spores was tested by inoculation of SCID mice. There was no marked loss of infectivity of spores stored at 4 degrees C for two years or frozen at -12 degrees C and -24 degrees C for 1, 8, and 24 h. Although there was a remarkable loss of infectivity, spores remained infective after freezing at -70 degrees C for 1 and 8 h. Heating at 60 degrees C and 70 degrees C for 5 min and 1 min, respectively, rendered the microsporidia non-infective. These findings demonstrate that E. cuniculi spores suspended in water can survive freezing temperatures but lost infectivity in water that reached a temperature of 60 degrees C at 5 min.