Several conditions of isolation were evaluated to determine which yielded the greatest number of thermotolerant and pathogenic freeliving amebae. Swab samples, easier to obtain and process, produced more pathogenic amebae than water samples. If water samples are required, 50-ml volumes gave the greatest percentage of pathogenic isolates. An incubating temperature of 42 degrees C yielded the most thermotolerant amebae. A total of 11 pathogenic isolates were obtained from 762 environmental samples and were Acanthamoeba (55%), Naegleria fowleri Carter, 1970 (27%), and N. australiensis De Jonckheere, 1981 (18%).