The Bohr protons released by oxygen exposure of the unliganded subunits of intermediates (alpha +CN-beta) (alpha +CN-beta) and (alpha beta +CN-) (alpha beta +CN-) were obtained by titrations of concentrated solutions of these species. The Bohr protons released by oxygen exposure of the other intermediates were obtained from titrations of equilibrium mixtures of two parental species, (alpha beta) (alpha beta), (alpha +CN-beta) (alpha +CN-beta), (alpha beta +CN-) (alpha beta +CN-), and (alpha +CN-beta +CN-) (alpha +CN-beta +CN-), in which the concentration of the hybrid intermediate was determined by cryogenic electrophoretic techniques. The Bohr effect of the intermediates was calculated by subtracting the Bohr protons released by oxygen exposure of the intermediates from the total Bohr protons of deoxyhemoglobin at the same pH. The Bohr effects of intermediates (alpha +CN-beta) (alpha beta) and (alpha beta +CN-) (alpha beta) were similar and vanished at pH 8 where the total Bohr effect of deoxyhemoglobin is still significant. This suggests that the Bohr effect in these intermediates is tertiary in the quaternary T structure. The curve of the Bohr effect of intermediate (alpha +CN-beta +CN-) (alpha beta), which was close to the curve obtained by adding the Bohr effects of the two monoliganded intermediates at acidic and physiological pH values, was significantly different from the curve obtained by adding the Bohr effects of one liganded subunit of intermediate (alpha +CN-beta) (alpha +CN-beta) and one liganded subunit of intermediate (alpha beta +CN-) (alpha beta +CN-).(ABSTRACT TRUNCATED AT 250 WORDS)