To determine how long spores of Encephalitozoon cuniculi, E. hellem, and E. intestinalis remain viable in seawater at environmental temperatures, culture-derived spores were stored in 10, 20, and 30 ppt artificial seawater at 10 and 20 C. At intervals of 1, 2, 4, 8, and 12 wk, spores were tested for infectivity in monolayer cultures of Madin Darby bovine kidney cells. Spores of E. hellem appeared the most robust, some remaining infectious in 30 ppt seawater at 10 C for 12 wk and in 30 ppt seawater at 20 C for 2 wk. Those of E. intestinalis were slightly less robust, remaining infectious in 30 ppt seawater at 10 and 20 C for 1 and 2 wk, respectively. Spores of E. cuniculi remained infectious in 10 ppt seawater at 10 and 20 C for 2 wk but not at higher salinities. These findings indicate that the spores of the 3 species of Encephalitozoon vary in their ability to remain viable when exposed to a conservative range of salinities and temperatures found in nature but, based strictly on salinity and temperature, can potentially remain infectious long enough to become widely dispersed in estuarine and coastal waters.