The role of internal and external reductants in the dopamine beta-monooxygenase (D beta M)-catalyzed conversion of dopamine to norepinephrine has been investigated in resealed chromaffin granule ghosts. The rate of norepinephrine production was not affected by the exclusion of internal ascorbate. The omission of ascorbate from the external medium drastically reduced the norepinephrine production without affecting the net rate of dopamine uptake. In the presence of the external reductant, the internal ascorbate levels were constant throughout the incubation period. The rate of norepinephrine production was not affected when ghosts were resealed to contain the D beta M reduction site inhibitor, imino-D-glucoascorbate. Ghosts incubated with external imino-D-glucoascorbate reduced the norepinephrine production. The weak D beta M reductant, 6-amino-L-ascorbic acid, was found to be a good external reductant for granule ghosts. The outcome of the above experiments was not altered when dopamine was replaced with the reductively inactive D beta M substrate, tyramine. These results and the known topology of membrane-bound D beta M disfavor the direct reduction of the enzyme by the external reductant. Our observations are consistent with the hypothesis that external ascorbate is the sole source of reducing equivalents for D beta M monooxygenation and that internal soluble ascorbate (or dopamine) may not directly reduce or mediate the reduction of membrane-bound D beta M in resealed granule ghosts.