The pH gradient (delta pH) across the membrane of bovine neurohypophysial granules was estimated by using the [14C]methylamine partition technique. The granule membrane showed high permeability to sugars such as sucrose or sorbitol and to cations. Granules suspended in sucrose medium showed an acidic internal delta pH that decreased with external pH from 1.0 pH unit at pH 7.5 to 0 at pH 4.5. High ionic strength of the external medium destroyed the delta pH, indicating that it originated from a Donnan equilibrium. In a medium constituted to simulate cytoplasm, the delta pH was approximately 0.5 pH unit. Aged neurosecretory granules were more acidic inside than were newly formed granules. The results are discussed in relationship to the nature and the duration of maturation and degradation process of the granule matrix.