Reversible monoamine oxidase A inhibitors (RIMA) are used as antidepressants but little is known about how they interact with the active site of the enzyme. Heterologous expression of human liver MAO-A in yeast provides sufficient protein for molecular studies and direct observation of the changes in the spectrum of the FAD co-factor when inhibitors bind. Using the reversible inhibitor, D-amphetamine, as a model compound, a concentration-dependent change in the spectrum with clean isosbestic points was observed. The decrease in absorbance between 400 and 500 nm gave a dissociation constant for binding similar to the K(i) value. Anaerobic reduction yielded the semiquinone spectrum only and the midpoint potential was the same as the free enzyme. Full reduction was not possible with dithionite as the reductant, suggesting that the semiquinone-reduced couple had a much lower midpoint potential than the free enzyme. In contrast, with substrate, which reduces the enzyme on an equimolar basis, the semiquinone is never seen. In anaerobic stopped-flow experiments, amphetamine inhibits completely the reoxidation of the reduced enzyme in contrast to a substrate such as 2-phenylethylamine (the desmethyl analogue of amphetamine) that accelerates the rate 12-fold. The spectral changes in MAO-A permit the examination of inhibitor interaction with the redox co-factor. Stacking of the inhibitor and flavin rings constitutes part of the interaction but, taking into account other evidence, steric factors may be the clue to the differences between substrate and inhibitor.