1-Phenylcyclopropylamine (1-PCPA) is shown to be an inactivator of the fungal flavoenzyme monoamine oxidase (MAO) N. Inactivation results in an increase in absorbance at 410 nm and is accompanied by the concomitant loss of the flavin absorption band at 458 nm. The spectral properties of the covalent adduct formed between the flavin cofactor of MAO N and 1-PCPA are similar to those reported for the irreversible inactivation product formed with I-PCPA and mammalian mitochondrial monoamine oxidase B [Silverman, R. B., and Zieske, P. A. (1985) Biochemistry 24, 2128-2138]. There is a hypsochromic shift of the 410 nm band upon lowering the pH to 2, indicating that an N5-flavin adduct formed upon inactivation. Use of the fungal enzyme, MAO N, which lacks the covalent attachment to the flavin adenine dinucleotide (FAD) cofactor present in the mammalian forms MAO A and MAO B, has allowed for the isolation and further structural identification of the flavin - inactivator adduct. The incorporation of two 13C labels into the inactivator, [2,3-13C2]-1-PCPA, followed by analysis using online liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy, provided a means to explore the structure of the flavin - inactivator adduct of MAO N. The spectral evidence supports covalent attachment of the 1-PCPA inactivator to the cofactor as N5-3-oxo-3-phenylpropyl-FAD.