Abstract An allosteric binding site with high affinity for imidazoline I 2 ligands has been proposed to exist on monoamine oxidase-B (MAO-B). However, enzyme inhibition only occurs at ligand concentrations far higher than are required to saturate this site. We here confirm previous reports that inactivation of recombinant human MAO-B with tranylcypromine results in the formation of a high affinity I 2 site on the enzyme, measured as an increase in binding of [ 3H]2-BFI. Incubation of MAO-B with 2-phenylethylamine, an endogenous trace amine and MAO-B substrate, resulted in a progressive loss of enzyme activity, increased enzyme mass, distinct spectral changes and, as was observed with tranylcypromine, a parallel increase in high affinity binding of [ 3H]2-BFI. Kinetic studies of the mechanism by which 2-BFI inhibits MAO-B activity suggested binding of 2-BFI, at micromolar concentrations, to a site distinct from the active site on at least two forms of the pure enzyme, probably corresponding to oxidised and reduced enzyme states. Studies with mutant enzymes revealed a pattern of changes consistent with binding of 2-BFI to the substrate entrance channel of human MAO-B. Structural data confirm that high affinity binding of I 2 ligands occurs within the entrance channel of inactive enzyme, while lower affinity binding at the same location in catalytically active enzyme results in mixed inhibition of MAO-B activity. High affinity I 2 sites may form in vivo due to inactivation of a portion of MAO-B during amine oxidation, while the low affinity I 2 site on active enzyme is a target for novel MAO-B inhibitor drugs.