ABSTRACT: I2-imidazoline receptors (I2-IR) are characterized by their high affinity for imidazolines and guanidines and medium affinity for imidazolidines. The differential recognition of I2-IR by amiloride led to subtype these sites as amiloride-sensitive (I2A -IR) and amiloride-insensitive (I2B-IR). I2-IR labeled with [3H] cbidazoxan or [3H]2-BFI in the rabbit cerebral cortex (I 2A -IR) displayed higher affinities for amiloride and amiloride analogs than in the rat cerebral cortex (I2B-IR). Other drugs tested displayed biphasic curves in competition experiments, indicating the existence of high and low affinity sites for both I2-IR subtypes. The drugs (+)- and (−)-medetomidine, bromoxidine, moxonidine, and clorgyline were more potent on the high and/or low affinity sites of I2B-IR than on I2A-IR. Preincubation (30 min at 25°C) with 10 −6 M isothiocyanatobenzyl imidazoline (IBI) or with 10−6 M clorgyline reduced by 40% and 26%, respectively, the binding of [3H]-BFI to I2B-IR, but it did not alter the binding of the radioligand to I2A-IR. These results indicated that the I2-IR subtypes differ in their pharmacologic profiles and in the nature of the imidazoline binding site involved in clorgyline and IBI alkylation. In rat cortical membranes, western blot detection of immunoreactive imidazoline receptor proteins revealed a double band of ∼29/30 kD and three less intense bands of ∼45, ∼66, and ∼85 kD. In rabbit cortical membranes the antibody detected proteins of ∼30, ∼57, ∼66, and ∼85 kD. It is suggested that I2-IR may be related to more than one receptor protein and that I2-IR subtypes differ in the nature of the proteins implicated.