Abstract The Bohr effect of bovine hemoglobin and of bovine hemoglobin in which protonic groups were substituted by reaction with FDNB was studied by means of normal, differential and difference titration curves. A number of results were obtained by comparison of calculated curves with the experimental curves. This study revealed the following facts. 1. 1. The presence of the normal and acid Bohr groups is easily seen from the differential titration curves of Hb and HbO 2 respectively. 2. 2. An analysis of these curves shows that only 18 of the 32 histidine residues are titratable and that 4 of these 18 residues have an abnormally high pK of about 8 in Hb as well as in HbO 2. 3. 3. From a comparison of the differential and difference titration curves of substituted hemoglobin with those of unsubstituted hemoglobin, it is concluded that 2 of the 4 α-amino groups are alkaline Bohr groups.