The distribution of nicotinic receptors in the brain and ganglia of the Cynomolgus monkey was studied by in situ hybridization and receptor autoradiography. A 35S-labeled antisense riboprobe for the mRNA of the alpha 3 subunit of the human nicotinic receptor, [3H]L-nicotine and alpha-bungarotoxin were used as markers. The highest levels of alpha 3-mRNA were observed in the hippocampus, the medial habenula, the lateral geniculate, the granular layer of the cerebellum, as well as in the pineal gland; moderate levels were found in other nuclei of the thalamus and in the deeper layers of the cerebral cortex. High-affinity binding sites for [3H]L-nicotine were observed mainly in the thalamus. The distribution of [125I]alpha-bungarotoxin binding sites was different from that observed for alpha 3-mRNA and [3H]L-nicotine; they were most abundant in a few specific thalamic nuclei, in the medial habenula and in lamina I of the cerebral cortex. The localization of these three markers was also investigated in the sympathetic, parasympathetic and sensory ganglia of the monkey. Intense labeling was observed for alpha 3-mRNA and for [125I]alpha-bungarotoxin in the sympathetic and parasympathetic ganglia, whereas no positive signal was seen in the ganglion of Gasser. [3H]L-nicotine binding was not detected in any of the ganglia examined. High levels of mRNA for the alpha 3 subunit of the nicotinic receptor were also detected in human sympathetic ganglia. Comparison between alpha 3-mRNA distribution and [3H]L-nicotine binding suggests that in the Cynomolgus monkey brain, the alpha 3 subunit may participate in the formation of more than one nicotinic receptor subtype: a high-affinity binding site for [3H]L-nicotine in the thalamus, and other sites with low affinity for nicotine in the medial habenula and cerebral cortex. Both the alpha 3-mRNA and the [125I]alpha-bungarotoxin are highly expressed in the sympathetic ganglia; however, since no information is presently available on the intraneuronal cellular localization, it cannot be established whether or not they are both present at synaptic sites.