The O₂ binding properties of bovine Hb were examined. The increase in Cl⁻ and DPG concentration enhanced P₅₀. A reduction in n_max was observed at high Cl⁻ concentration, while DPG had little effect on n_max. An increase in Cl⁻ concentration enhanced the Bohr effect, the magnitude of which reached a maximum at 0.1 M Cl⁻ and 20°C. This concentration is nearly equal to that at the highest slope of the log P₅₀ vs. log [Cl⁻] plot, and also equal to the physiological Cl⁻ concentration (0.1 M) of bovine blood. Furthermore, the influence of Cl⁻ concentration on the Bohr effect is independent of temperature. On the other hand, in the absence of Cl⁻, bovine Hb is sensitive to DPG; an increase in DPG concentration enhanced the Bohr effect, which reached a maximum at 3 mM DPG and 20°C. This concentration is nearly equal to that at the highest slope of the log P₅₀ vs. log [DPG] plot. At low DPG concentrations, the DPG effect on the Bohr effect became small with increasing temperature, whereas at high DPG concentrations, the DPG effect was insensitive to temperature changes. At the physiological concentration of DPG (0.5 mM), increases in both Cl⁻ concentration and temperature diminished the DPG effect. At the physiological concentrations of Cl⁻ and DPG, the Bohr effect was −0.36 at 37°C. The ΔH value at the physiological concentrations of Cl⁻ and DPG was approximately −5.8 kcal/mol at pH 7.4. These results indicate that Cl⁻ and temperature are important determinants of the O₂ binding properties of bovine Hb.