1. Intracellular hydrogen ion activity, [H+]i, was estimated in human erythrocytes and in nucleated avian erythrocytes from measurements of the distribution of ammonia and 5,5′-dimethyloxazolidine-2,4′-dione (DMO) between intracellular and extracellular fluid.
2. In human erythrocytes there was no difference between values for [H+]i derived from measurements of either DMO or ammonia.
3. In avian erythrocytes, [H+]i(ammonia) was consistently greater than [H+]i(DMO), indicating significant acid—base heterogeneity of the intracellular water. The degree of heterogeneity was assessed by reference to a theoretical model of two compartments of equal size.
4. Experiments with nuclei isolated from avian erythrocytes suggested that DMO is not bound to nucleoproteins, and that the nucleus may be more acidic than the cytoplasm.