Understanding the biochemical mechanisms that enable high-altitude animals to survive and function under conditions of hypoxic stress can provide important insights into the nature of physiological adaptation. Evidence from a number of high-altitude vertebrates indicates that modifications of hemoglobin function typically play a key role in mediating an adaptive response to chronic hypoxia. Because much is known about structure–function relationships of mammalian hemoglobins and their physiological role in oxygen transport, the study of hemoglobin variation in high-altitude mammals holds much promise for understanding the nature of adaptation to hypoxia from the level of blood biochemistry to the level of whole-organism physiology. In this review I 1st discuss basic biochemical principles of hemoglobin function and the nature of physiological adaptation to high-altitude hypoxia in mammals. I then discuss a case study involving a complex hemoglobin polymorphism in North American deer mice (Peromyscus maniculatus) that illustrates how integrative studies of protein function and fitness-related physiological performance can be used to obtain evolutionary insights into genetic mechanisms of adaptation.