The present investigation analyzes the cellular distribution of muscarinic and nicotinic acetylcholine receptors in rat neocortex, by use of monoclonal antibodies raised against purified receptor proteins. The degree of colocalization of both types of receptors was determined by way of immunofluorescent double-labeling techniques. For both classes of receptors, pyramidal and nonpyramidal cells were found immunostained and an identical laminar distribution pattern of immunopositive neurons in the rat neocortex became apparent. A striking similarity in distribution of the two cholinergic receptor types was found in the frontal/motor and parietal cortex. Accordingly, we observed a high degree of colocalization of muscarinic and nicotinic acetylcholine receptors within immunopositive cortical neurons. Approximately 90% of the cholinoceptive neurons expressed both types of receptors. The current data demonstrate that (i) the distribution of muscarinic and nicotinic cholinoceptive neurons in the neocortex is present in identical laminar patterns and represent the same type of cells, (ii) both classes of cholinergic receptors are highly colocalized within cholinoceptive neurons, which points at individual neurons as a likely site of interaction between muscarinic and nicotinic acetylcholine receptor-mediated processes.