The flavoprotein L-hydroxynicotine oxidase (LHNO) catalyzes an early step in the bacterial catabolism of nicotine. Although the structure of the enzyme establishes that it is a member of the monoamine oxidase family, LHNO is generally accepted to oxidize a carbon-carbon bond in the pyrrolidine ring of the substrate and has been proposed to catalyze the subsequent tautomerization and hydrolysis of the initial oxidation product to yield 6-hydroxypseudooxynicotine [Kachalova, G., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 4800-4805]. Analysis of the product of the enzyme from Arthrobacter nicotinovorans by nuclear magnetic resonance and continuous-flow mass spectrometry establishes that the enzyme catalyzes the oxidation of the pyrrolidine carbon-nitrogen bond, the expected reaction for a monoamine oxidase, and that hydrolysis of the amine to form 6-hydroxypseudooxynicotine is nonenzymatic. On the basis of the kcat/Km and kred values for (S)-hydroxynicotine and several analogues, the methyl group contributes only marginally (∼ 0.5 kcal/mol) to transition-state stabilization, while the hydroxyl oxygen and pyridyl nitrogen each contribute ∼ 4 kcal/mol. The small effects on activity of mutagenesis of His187, Glu300, or Tyr407 rule out catalytic roles for all three of these active-site residues.