Blood oxygen affinity and red blood cell properties were measured in three subspecies of genus Bufo: Bufo spinulosus limensis, collected at sea level and at an average day temperature of 20ºC; Bufo spinulosus trifolium, from 3100 m, average day temperature of 15ºC; and Bufo spinulosus flavolineatus, from 4100 m, average day temperature of 10ºC. Electrophoresis of the hemoglobin showed the same component in each of the three subspecies. At 20ºC the blood oxygen affinities (P50) showed small differences between Bufo spinulosus limensis and Bufo spinulosus trifolium, whereas the value for Bufo spinulosus flavolineatus was markedly lower. At 10ºC, the ambient temperature of Bufo spinulosus flavolineatus, the P50 was extremely low compared with the other two subspecies at their corresponding ambient temperatures