The biological action of flavoenzymes, such as flavin adenine dinucleotide (FAD)-containing monooxygenase, involves the formation of oxygenated flavin derivatives, such as 4 a-hydroperoxyflavin and 4 a-hydroxyflavin, in which a new center of chirality is created at the 4 a position during the enzymatic reactions. So far, the absolute configuration of this center of chirality in natural 4 a-oxygenated flavins has remained unknown in spite of its key importance for the diverse functions of flavoenzymes. Herein, we report the 4 a-hydroxy adduct 3 of 3-benzyl-5-ethyl-10-(tetraacetyl-D-ribityl)flavinium (1), one of the key intermediates involved in the enantioselective organocatalytic oxidation of sulfides to sulfoxides. The 4 a-hydroxyflavin diastereomers (+)-3 and (-)-3, separated by HPLC, were characterized by electronic circular dichroism (CD) spectroscopy. Their absolute configurations at the 4 a position were, for the first time, determined by comparing experimental CD spectra with those calculated by means of time-dependent density functional theory (TDDFT) on DFT-optimized structures obtained after an extensive conformation analysis.