L-Xyloascorbic acid (L-xylo-AsA) and its three stereoisomers, D-xyloascorbic acid (D-xylo-AsA), L-araboascorbic acid (L-arabo-AsA) and D-araboascorbic acid (D-arabc-AsA), have been considered to show some differences in vitamin C activity. In this paper the effect of L-xylo-AsA, D-xylo-AsA, L-arabo-AsA and D-arabo-AsA on the activity of dopamine beta-hydroxylase was studied to clarify whether or not the structural specificities of these stereoisomers have different effects on enzyme activity. The maximum velocity (Vmax) of the hydroxylation and Km for ascorbic acid were calculated using double-reciprocal plotting. Vmax for L-xylo-AsA was estimated to be 201 nmol/min/mg protein and those of D-xylo-AsA, L-arabo-AsA and D-arabo-AsA were 157 nmol/min/mg protein, 112 nmol/min/mg protein and 194 nmol/min/mg protein, respectively. Km for L-xylo-AsA was 1.5 mM and those for D-xylo-AsA, L-arabo-AsA and D-arabo-AsA were 2.3 mM, 2.7 mM and 1.4 mM, respectively. The effect of D-arabo-AsA on the activity of dopamine beta-hydroxylase was almost the same as that of L-xylo-AsA, while D-xylo-AsA and L-arabo-AsA showed smaller effects. Our results suggest that the configuration at carbon 4 might be more important than that of the hydroxyl group at carbon 5 for the development of the activity as a cofactor for dopamine beta-hydroxylase reaction.