Studies of abnormal and chemically modified haemoglobins indicate that in 0.1 M-NaCl about 40% of the alkaline Bohr effect of human haemoglobin is contributed by the C-terminal histidine HC3(146) beta. In deoxyhaemoglobin, the imidazole of this histidine forms a salt bridge with aspartate FG1(94) beta, in oxyhaemoglobin or carbonmonoxyhaemoglobin it accepts a hydrogen bond from its own NH group instead. Kilmartin et al. (1973) showed that in 0.2 M-NaCl + 0.2 M-phosphate this change of ligation lowered the pKa of the histidine from 8.0 in Hb to 7.1 in HbCO, but Russu et al. (1980) claimed that in bis-Tris buffer without added NaCl its pKa in HbCO dropped no lower than 7.85, and that in this medium the C-terminal histidine made only a negligible contribution to the alkaline Bohr effect. We have compared the histidine resonances of HbCO A with those of three abnormal haemoglobins: HbCO Cowtown (His HC3(146)beta----Leu), HbCO Wood (His FG4(97)beta----Leu) and HbCO Malmø (His FG4(97)beta----Gln). Our results show that the resonance assigned by Russu et al. to His HC3(146)beta in fact belongs to His FG4(97)beta. Although in Hb the pKa of His HC3(146)beta is 8.05 +/- 0.05 independent of ionic strength, in HbCO its pKa drops sharply with diminishing ionic strength, so that in the buffer employed by Russu et al. it has a pKa of 6.2 and makes a contribution to the alkaline Bohr effect that is 57% larger than in the phosphate buffer employed by Kilmartin et al. (1973). In HbCO A, His FG4(97)beta does not contribute to the Bohr effect, but in HbCO from which His HC3(146)beta has been cleaved (HbCO des-His), His FG4(97)beta is in equilibrium between two conformations with different pKa values. This equilibrium varies with ionic strength and pH, and presumably also with degree of ligation of the haem moiety. In HbCO A, His FG4(97)beta has a pKa of 7.8 compared to the pKa value of about 6.6 characteristic of free histidines at the surface of proteins. This high pKa is accounted for by its interaction with the negative pole at the C terminus of helices F and FG. It corresponds to a free energy change of the same order as that observed in the interaction of histidines with carboxylate ions and confirms the strongly dipolar character of alpha-helices, which manifests itself even when they lie on the surface of the protein.