Abstract The dissociation constant for the tetramer-dimer equilibrium of unliganded hemoglobin (Ku4,2) has been measured under a variety of solvent conditions by means of the carbon monoxide-binding technique described in the first paper in this series. Under standard conditions (0.1 m phosphate, pH 7.0) the dissociation equilibrium is characterized by the constant Ku4,2 = 3 x 10-12 m. Experiments in 0.5, 1.0, and 2.0 m NaCl indicate a continuous enhancement of dissociation by the salt to give values for Ku4,2 of 9 x 10-12 m, 18 x 10-12 m, and 65 x 10-12 m, respectively. The value of Ku4,2 also increases with pH in roughly the manner expected from considerations of the Bohr effect of hemoglobin. In contrast, dioxane up to 2 m has a slight stabilizing effect on the unliganded tetramer. These results are interpreted in terms of the salt bridges postulated to play a major role in the stability of unliganded hemoglobin tetramers and the cooperative interactions between hemoglobin and its ligands.