The distribution and properties of 125I-alpha bungarotoxin (125I-alpha BTX) binding in rat brain using micropunched tissue homogenates were examined with a binding technique. Highest level of 125I-alpha BTX binding was observed in the hypothalamus, followed by hippocampus, cortex, globus pallidus, nucleus caudatus and nucleus accumbens. Although high levels of 3H-quinuclidinyl benzilate (3H-QNB) binding in the striatum were found at dorso-lateral sites and low levels in the central regions, the level of 125I-alpha BTX binding within the striatum was almost equal. There was no significant correlation between 125I-alpha BTX binding and choline uptake which could be useful as a marker for functional cholinergic nerve terminals. The inhibition of specific 125I-alpha BTX binding to the hippocampal homogenates by nicotine, d-tubocurarine and other nicotine drugs was studied. The results of these studies suggest that alpha BTX binding sites in the brain are not likely the binding sites of nicotine or the physiological ACh R sites.