Three flavin-containing compounds, namely, riboflavin, riboflavin 5′-phosphate (flavin mononucleotide, FMN) and flavin-adenine dinucleotide (FAD, Scheme 1), were studied using matrix-assisted laser desorption ionization (MALDI), fast atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry. It was found that the isoalloxazine ring (flavin) was reduced in the positive-ion MALDI and FAB ionization processes producing unusual ions [M+2]^+· and [M+3]⁺ in addition to the anticipated [M+1]⁺, whereas ESI generated only [M+H]⁺ as the molecular-related ion. The reduction mechanism is hypothesized as the protonation from the matrix with a concomitant electron transfer,^*1 rather than the transfer of hydrogen atom(s), H^·_·ESI, in which matrix is not used, did not produce [M+2]^+· nor [M+3]⁺ more than the natural isotope abundance of ¹³C in the sample molecules. The reduction site expands to N(1)=C(10a)-C(4a)=N(5), a1,4-diaza-1,3-butadiene system, in the isoalloxazine ring, resembling the behavior of prosthetic groups of aerobic dehydrogenases.