To determine temperature tolerance of Echinostoma revolutum on the chick chorioallantois, 7-day-old preovigerous flukes were transferred to membranes for 7 days at 30, 32.5, 35, 37.5, 39, and 41 ? 1 C. Live flukes were recovered from the upper and lower surfaces of the chorioallantois, in the albumen, and on the surface of the embryo in eggs incubated at 30 to 39 C. At these temperatures chorioallantois-worms fed on blood. Histochemical tests for egg shell precursor material were positive in 32.5 to 39 C chorioallantois-worms. 35 to 39 C chorioallantoic-flukes were ovigerous, whereas those from the albumen were not. Eggs teased from 37.5 and 39 C flukes and incubated in tap water developed or hatched. Ovigerous worms varied considerably in size, some of which were not significantly larger than preovigerous or nonovigerous flukes. Information on temperature tolerance of trematodes of warm-blooded hosts is difficult to obtain from worms reared in definitive hosts. Transfer experiments have been used to determine survival of mammalian flukes in avian hosts. Thus Beaver (1937) transferred Echinostoma revolutum from the mammalian intestine to the cloaca of pigeons and found it was able to tolerate the higher temperature of the avian host. Studies on the survival and development of E. revalutum in normal or ectopic sites at temperatures below 37 C are not available. Recently Fried, Weaver, and Kramer (1968) cultivated this organism on the chick chorioallantois at 37 C to 38 C. Since the chorioallantois of 8to 12-day-old chick embryos can survive for approximately a week at 30 C (Fried, 1965; Fried and Weaver, 1969; Fried and Tomwall, 1969) a technique became available to study the lower range of temperature tolerance of E. revolutum. The purpose of this study was to determine survival and development of this fluke on the chick chorioallantois at 30 to 41 ? 1 C. MATERIALS AND METHODS Seven-day-old preovigerous worms obtained from laboratory-infected domestic chicks were transferred to chorioallantoic membranes (CAM) of 8to 9-day-old fertile white leghorn eggs as previously described for the cultivation of E. revolutum on the chick chorioallantois (Fried, Weaver, and Kramer, 1968). A total of 1,292 worms were placed on the membranes of 264 eggs, 2 to 10 per egg. Eggs incubated at 30, 32.5, 35, 37.5, 39, and 41 ? 1 C were examined 7 days postimplantation (Table I). To maintain some worms beyond Received for publication 3 June 1969 * Supported by NIH Grant AI-06835. 1 week at 30 C, 6 were transferred to new membranes, 2 per egg, for an additional 6 days. Eggs and worms were examined as described in the previous study. Seven-day-old worms and those grown on the chorioallantois for 7 days at 30 to 39 C were flattened on slides with moderate coverslip pressure, fixed in warm 70% ethanol, and stained with malachite green for basic proteins, Fast Red salt B for phenols, and catechol for polyphenol oxidase according to procedures described in Johri and Smyth (1956). The histochemical tests were used to assess development of flukes on membranes at different temperatures since Bell and Smyth (1958) and Smyth (1959) demonstrated that Diplostomum phoxini cultivated under suboptimal conditions showed deficiencies in egg shell precursor material. Length and width measurements were made on fixed worms (Table II). To determine their viability, eggs teased from the uteri of worms grown on the chorioallantois at 35, 37.5, and 39 C were incubated in tap water at 30 C for up to 15 days. Fifty 7-day-old worms maintained 2 per petri dish in 15 ml of nonnutrient Tyrode's solution containing 200 units penicillin and 1,000 ~g streptomycin/ml and incubated at 30 to 39 C were used to determine survival of E. revolutum in Tyrode's.