Bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the copper-, ascorbate-, and O2-dependent cleavage of C-terminal glycine-extended peptides, N-acylglycines, and the bile acid glycine conjugates to the corresponding amides and glyoxylate. Two known metabolites of aspirin, salicyluric acid and gentisuric acid, are also substrates for PAM, leading to the formation of salicylamide and gentisamide. The time course for O2 consumption and glyoxylate production indicates that salicylurate amidation is a two-step reaction. Salicylurate is first converted to N-salicyl-alpha-hydroxyglycine, which is ultimately dealkylated to salicylamide and glyoxylate. The enzymatically generated salicylamide and N-salicyl-alpha-hydroxyglycine were characterized by mass spectrometry and two-dimensional 1H-13C heteronuclear multiple quantum coherence NMR.