Pythium insidiosum causes a potentially life-threatening infectious disease called pythiosis. An early, accurate diagnosis is important, since prompt treatment leads to a better prognosis. Unsuccessful attempts to isolate the organism have been associated with specimens subjected to lower temperatures. We analyzed growth of P. insidiosum at various temperatures. Culture at low (8 degrees C) and high (42 degrees C) temperatures resulted in death or inhibited growth of the organism. Culture under optimal temperatures (28 and 32 degrees C) was important for successful isolation of P. insidiosum.