There is a great deal of interest in the role of nicotinic acetylcholine receptors in the central nervous system, although their function is not well understood at present. Currently, central nicotinic receptors can be classified broadly as either alpha-bungarotoxin binding sites with low affinity for acetylcholine agonists, or as high-affinity agonist binding sites with low affinity for alpha-bungarotoxin. Neuronal nicotinic receptors with a high affinity for agonists are distributed widely in the central nervous system. Evidence from molecular biology and electrophysiology suggests that multiple nicotinic receptor types exist in the brain. In this study we have used the agonist [3H]cytisine as a ligand for autoradiography to generate a detailed quantitative map of the high-affinity agonist binding nicotinic receptor in the rat brain. Optimized binding conditions, characterization of the kinetic and equilibrium binding properties, and demonstration of the nicotinic pharmacology of this binding site in tissue sections confirm the usefulness of [3H]Cytisine as a ligand for nicotinic receptor autoradiography. [3H]Cytisine autoradiography provides excellent anatomic resolution with very low non-specific binding. This property has allowed us to describe variations in receptor density within subnuclei and gradients of receptor density in larger brain regions. Data from several studies suggest that the predominant high-affinity agonist binding nicotine receptor in the central nervous system is composed of the alpha 4 and beta 2 subunits. The data in the current study are consistent with the suggestion that [3H]cytisine labels only the alpha 4 beta 2 nicotinic receptor with high affinity, offering the possibility of localizing a specific nicotinic receptor subtype in the central nervous system. In summary, we characterize the optimum experimental conditions for the use of [3H]cytisine in tissue section autoradiography. [3H]Cytisine proves to be an excellent marker for nicotinic cholinergic receptors with a very high affinity and very low background. We provide a detailed quantitative characterization of nicotinic receptor density in the rat central nervous system and we find there are significant variations and gradients in receptor density within specific brain regions, including subregions previously thought to be homogeneous.