The in vitro autoradiographic technique was used to characterize the density and laminar distribution of muscarinic cholinergic receptors in 12 cytoarchitectonic areas in the frontal, parietal, and occipital lobes of the rhesus monkey. The entire population of muscarinic receptors was labeled with [3H]quinuclidinyl-benzilate; the M1 receptor subtype was labeled with [3H]pirenzepine; and the density of the M2 receptor subtype was estimated by subtracting the density of M1 receptors from the total population. The overall density of M1 and M2 receptor subtypes was similar throughout the cerebral cortex. However, their laminar distribution varied regionally. In cortical regions of the parietal and occipital lobes and in the primary motor cortex of the frontal lobe, both M1 and M2 receptor subtypes were concentrated in the supragranular layers. By contrast, in prefrontal cortical areas, the combined population of M1 and M2 receptors was evenly distributed across the cortical layers, though M1 receptors were most dense and M2 receptors least dense in layer IV. The difference in the distribution of cholinergic receptors in the prefrontal cortex compared to other neocortical areas reveals a degree of chemoarchitectural specificity of this region with respect to cholinergic markers that has escaped immunohistochemical and other anatomical and functional techniques.