Cortical neurons immunoreactive to nicotinic acetylcholine receptors (nAChR) of the rat brain were characterized with monoclonal antibodies directed to ACh-binding subunits (alpha 4) or to ACh-structural subunits (beta 2). A heterogeneous population of nAChR-LI neurons was found in all cortical regions. The most prominent immunoreactive neurons were pyramids of layers V and II-III. The nonpyramidal positive neurons were fusiform horizontally oriented neurons of layer VIb, small cells of layer I and round or ovoid neurons of layers II-V. Double labeled experiments (immunohistochemistry and fluorescent retrograde tracers) showed that cholinoceptive pyramidal neurons of layer V project mainly to subcortical targets such as caudate-putamen, superior colliculus, and pontine nuclei, while very few nAChR positive neurons connect to other cortical areas. These findings suggest that the mainly excitatory effect that has been attributed to the cholinergic innervation upon the cortical neurons may have a greater influence upon the cortico-subcortical output than the corticortical one.