To date there is ample evidence that patients with vitiligo accumulate millimolar concentrations of hydrogen peroxide (H2O2) in their epidermis as well as in their blood lymphocytes/monocytes. Several enzymes are affected by this H2O2 including catalase, glutathione peroxidase, and 4 alpha-carbinolamine dehydratase. The latter enzyme disrupts the recycling of the essential cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH4) for the aromatic amino acid hydroxylases as well as the nitric oxide synthases. In this report we have elucidated the influence of H2O2 on dihydropteridine reductase (DHPR), the last enzyme in the 6BH4-recycling process. Here we show for the first time that concentrations of less than 30 microM H2O2 increase DHPR activities, whereas levels greater than 30 microM H2O2 deactivate the enzyme based on the oxidation of Met146 and Met151 in the sequence, consequently leading to disruption of the NADH-dependent enzyme active site. This oxidation was confirmed by Fourier transform-Raman spectroscopy yielding the expected SO band at 1025 cm-1 characteristic of methionine sulfoxide. Hence these results unmasked a novel regulatory mechanism for DHPR enzyme activity. Moreover, we also demonstrated that DHPR activities in whole blood of patients with vitiligo are significantly decreased in untreated patients, whereas activities are normalized after removal of epidermal H2O2 with a topical pseudocatalase (PC-KUS). Taken together, these new data add more evidence to a systemic involvement of H2O2 in the pathomechanism of vitiligo.