Whereas it is widely believed that animals native to high altitude show lower O2 partial pressures at 50% hemoglobin saturation (P50) than do related animals native to low altitude, that “fact” has not been well documented. Consequently, P50 at pH 7.4, PCO2(7.4), the CO2 Bohr effect, and the buffer slope (delta log PCO2/delta pH) were determined via the mixing technique in Peromyscus maniculatus native to a range of altitudes but acclimated to 340 or 3,800 m. PCO2(7.4) and buffer slope were substantially lower at high altitude. The change in P50(7.4) between acclimation altitudes was minimal (0.8% increase at 3,800 m), because of counterbalancing changes in PCO2, 2,3-diphospho-D-glycerate concentration, and perhaps other factors. At both acclimation altitudes there was a highly significant negative correlation between P50(7.4) and native altitude. Since pH in vivo probably increases slightly at high altitude, the data on P50 corrected to pH 7.4 are probably underestimates of the difference in in vivo P50 at low vs. high altitude. Hence these results corroborate theoretical predictions that low P50 is advantageous under severe hypoxic stress.