Two angiotensin II receptor subtypes were distinguished in the rat brain using in vitro receptor autoradiography based on the differential effects of sulfhydryl reducing agents on 125I-sarcosine1, isoleucine8 angiotensin II binding in various brain nuclei. At several nuclei, e.g. the hypothalamus, circumventricular organs and the dorsal medulla, 125I-sarcosine1, isoleucine8 angiotensin II binding was strongly inhibited by 30 mM β-mercaptoethanol or 5 mM dithiothreitol, whereas at other nuclei, e.g. the lateral septum, colliculi, locus coeruleus and medial amygdala, sulfhydryl reducing agents had either little effect on radioligand binding or enhanced the binding. The distribution of the sulfhydryl reducing agent inactivated subtype corresponds exactly with the distribution of DuP 753 sensitive (designated as AIIα) 125I-sarcosine1, isoleucine8 angiotensin II binding sites25. The subtype not inhibited by sulfhydryl reducing agents corresponds with the DuP 753 insensitive (designated as AIIβ) sites in the brain25. The sulfhydryl reducing agent effect on brain angiotensin II receptor subtypes is similar to that seen in angiotensin II receptor subtypes in peripheral tissues. These observations indicate that many previous studies of brain angiotensin II receptor binding that included 5 mM dithiothreitol in the assay medium overlooked the sulfhydryl reducing agent inactivated (AIIα) receptor subtype.