When freshwater turtles acclimatize to winter hibernation, there is a gradual transition from aerobic to anaerobic metabolism, which may require adjustments of blood O₂ transport before turtles become anoxic. Here we report the effects of protons, anionic cofactors and temperature on the O₂-binding properties of isolated hemoglobin (Hb) isoforms, HbA and HbD, in the turtle Trachemys scripta. We determined the primary structures of the constituent subunits of the two Hb isoforms, and we related the measured functional properties to differences in O₂ affinity between untreated hemolysates from turtles that were acclimated to normoxia and anoxia. Our data show that HbD has a consistently higher O₂ affinity compared to HbA, whereas Bohr and temperature effects as well as thiol reactivity are similar. Although sequence data show amino acid substitutions at two known β-chain ATP-binding site positions, we find high ATP affinities for both Hb isoforms, suggesting an alternative and stronger binding site for ATP. The high ATP affinities indicate that, although ATP levels decrease in red blood cells of turtles acclimating to anoxia, the O₂ affinity would remain largely unchanged, as confirmed by O₂-binding measurements of untreated hemolysates from normoxic and anoxic turtles. Thus, the increase in blood-O2 affinity that accompanies winter acclimation is mainly attributable to a decrease in temperature rather than in concentrations of organic phosphates. This is the first extensive study on freshwater turtle Hb isoforms providing molecular evidence for adaptive changes in O₂ transport associated with acclimation to severe hypoxia.