[3H]-Methyllycaconitine ([3H]-MLA) is a new radioligand with selectivity for α7-type neuronal nicotinic acetylcholine receptors (nAChRs). In our previous study [Davies, A.R.L., Hardick, D.J., Blagbrough, I.S., Potter, B.V.L., Wolstenholme, A.J. & Wonnacott, S. (1999) Neuropharmacology, 38, 679–690], this radioligand labelled a single class of site in rat brain membranes; its pharmacology and distribution in crudely dissected brain regions closely paralleled that of the well-established α7-ligand [125I]-α-bungarotoxin. However, a small population of [3H]-MLA binding sites was apparently insensitive to α-bungarotoxin. Here we have extended the study to mouse brain, using autoradiography to examine the distribution of [3H]-MLA and [125I]-α-bungarotoxin binding sites. [3H]-MLA labelled a single class of site in mouse brain membranes with a KD of 2.2 nm and a Bmax of 45.6 fmol/mg protein. Specific binding, defined by unlabelled MLA (Ki = 0.69 nm), was completely inhibited by (–)-nicotine (Ki = 1.62 μm), whereas α-bungarotoxin inhibited only 85% of specific binding (Ki = 3.5 nm). The distributions of [125I]-α-bungarotoxin and [3H]-MLA binding sites were compared by autoradiography, and binding was quantitated in 72 brain regions. Binding of both radioligands was highly correlated, with highest densities in the dorsal tegmental nucleus of the pons, colliculi and hippocampus. Serial sections labelled with [3H]-MLA in the absence or presence of unlabelled MLA or α-bungarotoxin provided no evidence for any α-bungarotoxin-resistant binding. The results are discussed in terms of binding sites that are inaccessible to α-bungarotoxin in membrane preparations. This study demonstrates the utility of [3H]-MLA for characterization of α7-type nicotinic receptors in mammalian brain, and suggests that it labels a population identical to that defined by [125I]-α-bungarotoxin.