Imidazoline binding sites (IBS) were proposed to be responsible for some of the pharmacological and therapeutic activities of imidazoline and related compounds and have been classified into two subtypes, I1BS and I2BS. Convergent studies attribute a role in central blood pressure regulation to the I1BS. In contrast, the function of I2BS remains unknown. In the present study, by combining biochemical and molecular biology approaches, we show that 1) microsequencing of I2BS purified from rabbit kidney mitochondria allowed the recovery of four peptide sequence stretches displaying up to 85.7% similarity with human, rat, and bovine monoamine oxidases (MAO)-A and -B; 2) I2BS and MAO displayed identical biophysical characteristics as their activities, measured by [3H]idazoxan binding and [14C]tyramine oxidation, respectively, could not be separated using various chromatographic procedures; and 3) heterologous expression of human placenta MAO-A and human liver MAO-B in yeast, inherently devoid of I2BS and MAO activities, led to the coexpression of [3H]idazoxan binding sites displaying ligand-recognition properties typical of I2BS. These results show definitely that I2BS is located on both MAO-A and -B. The fact that I2BS ligands inhibited MAO activity independently of the interaction with the catalytic region suggests that I2BS might be a previously unknown MAO regulatory site.