High affinity and temperature sensitivity of blood oxygen binding in Pangasianodon hypophthalmus due to lack of chloride- hemoglobin allosteric interaction. Am J Physiol Regul Integr Comp Physiol 308: R907–R915, 2015. First published March 25, 2015; doi:10.1152/ajpregu.00470.2014.—Air-breathing fishes represent in- teresting organisms in terms of understanding the physiological changes associated with the terrestrialization of vertebrates, and, further, are of great socio-economic importance for aquaculture in Southeast Asia. To understand how environmental factors, such as high temperature, affect O2 transport in air-breathing fishes, this study assessed the effects of temperature on O2 binding of blood and Hb in the economically important air-breathing fish Pangasianodon hypo- phthalmus. To determine blood O2 binding properties, blood was drawn from resting cannulated fishes and O2 binding curves made at 25°C and 35°C. To determine the allosteric regulation and thermody- namics of Hb O2 binding, Hb was purified, and O2 equilibria were recorded at five temperatures in the absence and presence of ATP and Cl. Whole blood had a high O2 affinity (O2 tension at half saturation P50 4.6 mmHg at extracellular pH 7.6 and 25°C), a high temper- ature sensitivity of O2 binding (apparent heat of oxygenation Happ 28.3 kcal/mol), and lacked a Root effect. Further, the data on Hb revealed weak ATP binding and a complete lack of Clbinding to Hb, which, in part, explains the high O2 affinity and high temperature sensitivity of blood O2 binding. This study demonstrates how a potent mechanism for increasing O2 affinity is linked to increased tempera- ture sensitivity of O2 transport and provides a basic framework for a better understanding of how hypoxia-adapted species will react to increasing temperatures.