Our experiments revealed that Bohr factors for the high- and low-altitude Hb variants are very similar in spite of the differences in O2-affinity. The Bohr factors of deer mouse Hbs are also comparable to those of other mammalian Hbs. In contrast, the high- and low-altitude variants of deer mouse Hb exhibited similarly low temperature sensitivities that were independent of red blood cell anionic cofactors, suggesting an appreciable endothermic allosteric transition upon oxygenation. In conclusion, high-altitude deer mice have evolved an adaptive increase in Hb–O2 affinity, but this is not associated with compensatory changes in sensitivity to changes in pH or temperature. Instead, it appears that the elevated Hb–O2 affinity in high-altitude deer mice is compensated by an associated increase in the tissue diffusion capacity of O2 (via increased muscle capillarization), which promotes O2 unloading.