BU99006 is an irreversible I2 ligand which selectively inactivates I2 binding sites, making it an ideal tool with which to study I2 site mechanism. We sought to determine the effects of BU99006 on I2 binding in relation to monoamine oxidase (MAO), and the time course of these effects. In vitro, rat brain membranes that were pre-treated with 10 μM BU99006 showed no change in MAO activity, despite suffering a significant reduction in [3H]2BFI binding (52.5 ± 19.6 to 8.5 ± 3.8 fmol mg−1, 84%). Furthermore, reversible I2 ligands 2BFI and BU224 were able to inhibit MAO, whether treated with BU99006 or not. In vivo, a 5 mg kg−1 i.v. dose of BU99006 in rats rapidly reduced [3H]2BFI binding with similar magnitude (85%, maximal reduction after 20 min), without effect on either MAO activity or the specific binding of selective MAO-A and MAO-B radioligands. Moreover, following this irreversible treatment, recovery of central [3H]2BFI binding occurred with a rapid half-life of 4.3 h in rat brain (2.0 h in mouse), which is not consistent with a site on MAO. These data indicate that the high affinity site which is occupied by [3H]2BFI and irreversibly binds BU99006, is not the same as that which causes inhibition of MAO, and may point to the existence of another I2 binding site.