The role of histidine (HC3) 146 beta in the previously established pH-dependent properties of the R state of human hemoglobin has been investigated. The rate constants for the dissociation and combination of the fourth carbon monoxide molecule, l4 and l'4, have been determined as a function of pH for hemoglobin A and des-(His 146 beta) hemoglobin A. From these kinetic parameters, the value of L4, the affinity constant for the 4th carbon monoxide molecules, has been calculated according to the equation L4 = l'4/l4. In addition, the effect of removal of histidine 146 beta or its replacement by arginine (hemoglobin Cochin-Port Royal) on k4, the rate of oxygen dissociation from fully liganded hemoglobin, has been determined as a function of pH. Removal of histidine 146 beta reduces the pH dependence of L4 by 47%. At the same time, it produces a similar, 45%, reduction in the pH dependence of kr. Replacement of histidine 146 beta by arginine reduces the pH dependence of k4 by 23% and that of l'4 by about 30%. These chemical modifications cause reductions in the R state Bohr effect which are remarkably similar in magnitude to the reduction which they produce in the overall Bohr effect. These results indicate that histidine 146 beta controls a major fraction of the R state Bohr effect as well as being a major participant in the overall and T state Bohr effects.