Monoamine oxidases (MAO) A and B are approximately 60-kDa outer mitochondrial membrane flavoenzymes catalyzing the degradation of neurotransmitters and xenobiotic arylalkyl amines. Despite 70% identity of their amino acid sequences, both enzymes exhibit strikingly different properties when exposed to thiol-modifying reagents. Human MAO A and MAO B each contain 9 cysteine residues (7 in conserved sequence locations). MAO A is inactivated by N-ethylmaleimide (NEM) much faster (tau(1/2) = approximately 3 min) than MAO B (tau(1/2) = approximately 8 h). These differences in thiol reactivities are also demonstrated by monitoring the NEM modification stoichiometries by electrospray mass spectrometry. Inactivation of either enzyme with acetylenic inhibitors results in alterations of their thiol reactivities. Cys5 and Cys266 were identified as the only residues modified by biotin-derivatized NEM in clorgyline-inactivated MAO A and pargyline-inactivated MAO B, respectively. The x-ray structure of MAO B (Binda, C., Newton-Vinson, P., Hubalek, F., Edmondson, D. E., and Mattevi, A. (2002) Nat. Struct. Biol. 9, 22-26) shows that Cys5 is located on the surface of the molecule opposite to the membrane-binding region. Cys266 in MAO A is predicted to be located in the same region of the molecule. These thiol residues are also modified by biotin-derivatized NEM in the mitochondrial membrane-bound MAO A and MAO B. This study shows that the MAO A structure is "more flexible" than that of MAO B and that clorgyline and pargyline inactivation of MAO A and B, respectively, increases the structural stability of both enzymes. No evidence is found for the presence of disulfide bonds in either enzyme, contrary to a previous suggestion.