Cholinergic nicotinic receptors in rat brain were demonstrated by the use of the potent nicotinic antagonist [125I]alpha-bungarotoxin [125I]alpha-Btx). Biochemical studies on binding of [125I]alpha-Btx to rat hippocampal homogenates revealed saturable binding sites which are protected by nicotine, D-tuborcurarine and acetylcholine but not by atropine or oxotremorine. The hippocampus and hypothalamus displayed relatively high [125I]alpha-Btx specific binding whereas the cerebellum was devoid of specific binding. Other regions displayed intermediate binding levels. Analysis of the regional distribution of [125I]alpha-Btx binding by autoradiography of frontal brain sections revealed high labeling in the hippocampus, hypothalamic supraoptic, suprachiasmatic and periventricular nuclei, ventral lateral geniculate and the mesencephalic dorsal tegmental nucleus. It is suggested that the limbic forebrain and midbrain structures as well as sensory nuclei are the main nicotinic cholinoceptive structures in the brain.