The distribution of acetylcholinesterase-rich axons was compared to that of choline acetyltransferase-positive (cholinergic) axons in 28 major cytoarchitectonic divisions of the adult human cerebral cortex. Acetylcholinesterase-rich as well as choline acetyltransferase-positive cortical axons contained multiple varicosities. Each type of axon was more densely distributed in limbic-paralimbic regions of the brain. In all the cortical areas that were examined, the two markers displayed laminar and regional distribution patterns that were indistinguishable from each other. A method that allowed the concurrent visualization of both reaction products demonstrated that acetylcholinesterase and choline acetyltransferase were colocalized in the same axon. These observations show that there is probably a complete correspondence between choline acetyltransferase-positive and acetylcholinesterase-rich axons and that the acetylcholinesterase reaction can be used as a specific marker for cortical cholinergic axons in the adult human brain.