This is a follow-up report on the viability of pathogenic Naegleria fowleri, Naegleria australiensis and Acanthamoeba castellanii isolates during 5 to 10 years of cryopreservation at –70°C. The greatest decrease in viability occurred with N. fowleri and the least occurred with N. australiensis. At 10 years of cryostorage, viability was 21% for N. fowleri, 32% for A. castellanii and 51% for N. australiensis.