The radiolabeled angiotensin II (ANG II) antagonist, [125I]-sar1, ile8-ANG II, was used to study brain ANG II receptors by both homogenate binding and in vitro autoradiography. In homogenate preparations of the hypothalamus, thalamus, septum and midbrain (HTSM), [125I]-sar1 ile8-ANG II bound to a single class (Hill slope 0.84 ± 0.05) of high affinity binding sites (KD 0.42 ± 0.03 nM, Bmax 5.98 ± fmol/mg protein). Competition for the [125I]-sar1 ile8-ANG II binding site in HTSM membranes demonstrated a rank order potency characteristic of binding to the ANG II receptor, with the unlabeled antagonist being slightly more potent than ANG II (Ki 0.22 ± 0.03 vs. 0.95 ± 0.06 nM, respectively). Brain slices from the region of the rostral third ventricle were incubated with 0.5 nM [125I]-sar1, ile8-ANG II in the presence or absence of 1 µM ANG II and exposed to LKB Ultrofilm. Autoradiographic images of [125I]-sar’, ile8-ANG II binding revealed that structures situated within the anterior wall of the third ventricle, i.e. the lamina terminalis, were heavily labeled; including the subfornical organ, median preoptic nucleus and organum vasculosum laminae terminalis. These results show the utility of [125I]-sar1, ile8-ANG II as a probe to study brain ANG II receptors and provides pharmacological evidence for the rostral third ventricle as a possible site for central ANG II