In contrast to birds and mammals, no information appears to be available on the molecular adaptations for O2 transport in high-altitude ectothermic vertebrates. We investigated Hb of the aquatic Andean frog Telmatobius peruvianus from 3,800-m altitude as regards isoform differentiation, sensitivity to allosteric cofactors, and primary structures of the ␣-and -chains, and we carried out comparative O2-binding measurements on Hb of lowland Xenopus laevis. The three T. peruvianus isoHbs show similar functional properties. The high O2 affinity of the major component results from an almost complete obliteration of chloride sensitivity, which correlates with two ␣-chain modifications: blockage of the NH2-terminal residues and replacement by nonpolar Ala of polar residues Ser and Thr found at position ␣131(H14) in human and X. leavis Hbs, respectively. The data indicate adaptive significance of ␣-chain chloride-binding sites in amphibians, in contrast to human Hb where chloride appears mainly to bind in the cavity between the -chains. The findings are discussed in relation to other strategies for high-altitude adaptations in amphibians.amphibians; chloride binding; hypoxia; organic phosphates; oxygen transport HOW IS OXYGEN TRANSPORT to metabolizing tissues secured at high altitude? In contrast to intensive investigations in birds and mammals (7,39,59), the molecular strategies for O 2 transport in high-altitude ectothermic vertebrates remain unexplored, despite greater variations in environmental conditions (temperature, pH, O 2 tension, etc.) and lesser capacities for homeostatic regulation of internal physical and chemical conditions compared with homeothermic vertebrates and a long-standing interest in high-altitude aquatic amphibians (1, 24).The anuran genus Telmatobius (that variously is referred to as frogs or toads) occurs in the Andes mountains at altitudes from 2,000 to over 4,000 m (14) where aerial O 2 tensions fall from ϳ159 mmHg at sea level to ϳ92 mmHg. The hypoxic stress is compounded in aquatic species, particularly at night when photosynthetic activity in the ponds ceases (21). T. culeus found in Lake Titicaca at 3,812 m has reduced, poorly developed lungs but exhibits compensatory physiological and behavioral adaptations (24) that include an "oversized," folded skin, which is penetrated by cutaneous capillaries and ventilated by "bobbing" behavior under hypoxia, and small erythrocytes and higher erythrocyte counts, blood-O 2 affinities, and O 2 -carrying capacities than anurans living at sea level (24). Subspecies of Bufo spinulosus living at sea level and at 3,100 to 4,100 m in the Andes analogously exhibit increasing blood-O 2 affinities with altitude (43).The O 2 affinity of blood is a product of the intrinsic O 2 affinity of the Hb molecules and the erythrocytic effectors that modulate Hb-O 2 affinity. Compared with mammals that use 2,3-diphosphoglycerate (DPG) and fish that use ATP (often in conjunction with the more potent effector guanosine triphosphate) (60) as organic O 2 -affinity modulat...