A series of ascorbate derivatives has been used to examine the specificity and the chemistry of the reduction site of dopamine beta-monooxygenase (D beta M). Replacement of the 6-OH group of ascorbic acid with either bromine or hydrogen does not alter the enzyme reduction efficiency significantly. Unexpectedly, the 6-OH modified ascorbate derivatives, 6-S-phenyl-6-thio-L-ascorbic acid and 6-O-phenyl-L-ascorbic acid were found to have much higher affinity for the enzyme than the most effective known electron donor, ascorbic acid (AscH-). The affinity of 2-amino-6-S-phenyl-L-ascorbic acid was found to be similar to that of 2-amino-L-ascorbic acid. 6-Amino-6-deoxy-L-ascorbic acid is neither a substrate nor an inhibitor for the enzyme. Although glucoascorbic acid is an excellent substrate for the enzyme, imino glucoascorbic acid was found to be an extremely potent competitive inhibitor for the enzyme. The stereoelectronic properties and alternate binding modes of these molecules have been considered in explaining the observations.