Pharmacologically active compounds with an imidazoline and/or guanidinium moiety are recognized with high affinity by a family of membrane-bound proteins collectively known as imidazoline binding sites or imidazoline/guanidinium receptive sites. Two such receptive sites may correspond to imidazoline binding domains identified on the A and B isoforms of monoamine oxidase (MAO), but the detection of monoamine oxidase isoforms in multiple tissues contrasts with the restricted expression of imidazoline-binding proteins. To address these issues, we determined the relationship between monoamine oxidase isoforms and subtypes of imidazoline-binding proteins in human tissues known to express one or both isoforms of MAO. 2-(3-Azido-4-[125I]iodophenoxy)methylimidazoline ([125I]A-ZIPI), a photoaffinity adduct that selectively labels imidazoline-binding proteins, photolabeled an M(r) = approximately 59,000 peptide in liver and an M(r) = approximately 63,000 peptide in placenta, consistent with the M(r) of the MAO isoforms identified by immunoblots in these tissues. The photolabeled species in liver was immunoprecipitated with MAO-B selective antibodies, whereas the photolabeled species in placenta was immunoprecipitated by MAO-A selective antibodies consistent with the isoform of MAO predominantly expressed in these tissues. The imidazoline/guanidinium ligands interact with the enzyme at a site distinct from the substrate recognition domain, and the immunoprecipitated peptides in liver and placenta display distinct ligand recognition properties consistent with those reported for subtypes of imidazoline binding sites. However, the imidazoline binding domain was not detected in platelet membrane preparations containing amounts of MAO-B equivalent to those in the photolabeled liver membranes indicating that recognition of this domain is tissue-restricted. Restricted access to the imidazoline binding domain on platelet MAO-B was not altered by membrane washing with 500 mM KCl or by solubilization and partial purification of the enzyme suggesting that there are distinct subpopulations of MAO. Identification of a binding domain on MAO that recognizes this class of pharmacologically active compounds suggests a novel mechanism for regulation of substrate oxidation/selectivity or that the enzyme may subserve an as yet undefined function.