Blastocystis infection in amphibians was surveyed in three species of anuran and one species of urodele amphibians captured at two distinct locations in Japan. All three species of frogs were highly infected with Blastocystis, while 69 individual urodele newts, Cynopus pyrrhogaster, were negative for infection. Eleven Blastocystis isolates (47.8%) were recovered from 23 Rana nigromaculata leopard frogs. Twenty-three (92%) of 25 Rana catesbeiana bullfrogs and all (100%) of 24 Bufo japonicus japonicus toads were positive for Blastocystis. Two distinct populations of the toad and bullfrog showed a high prevalence (100 or 84.6%) of Blastocystis infection, while in two populations of the leopard frog only one population was positive for Blastocystis (84.6%). Three Blastocystis isolates from different species of the frogs were established. Since none of the three isolates could survive at 37 degrees C, a temperature tolerance assay was performed to assess the optimal growth temperature and to determine the range of non-lethal temperatures. During the exponential growth phase of 3- or 4-day cultures at 25 degrees C, three isolates were exposed to 4, 28, 31, or 34 degrees C for 3 days and then returned to 25 degrees C to monitor the cell growth. Based on the optimal growth temperatures and different ranges of temperature tolerance among the three new isolates from frogs and two known species, Blastocystis hominis and Blastocystis lapemi, it was established that the three isolates recovered from different species of frogs had different physiological features from B. hominis and B. lapemi.