Parkinson's disease is a neurodegenerative disorder characterised by motor and non-motor impairments, including constipation. The hallmark pathological features of Parkinson's disease are Lewy bodies and neurites, of which aggregated α-synuclein is a major constituent. Frequently, Lewy pathology is identified in the distal gut of constipated Parkinson's disease patients. The neurons that innervate the distal gut that express α-synuclein have not been identified. We used multiple-labeling immunohistochemistry and anterograde tracing to quantify which neurons projecting to the guinea-pig rectum and human colon expressed α-synuclein in their axons. α-Synuclein-immunoreactivity was present in 24 ± 0.7% of somatostatin (SOM)-immunoreactive (IR) varicosities; 20 ± 4.3% of substance P (SP)-IR varicosities and 9 ± 1.3% vasoactive intestinal polypeptide (VIP)-IR varicosities in guinea-pig rectal myenteric ganglia. However, α-synuclein-immunoreactivity was localised in significantly more vesicular acetylcholine transporter (VAChT)-IR varicosities (88 ± 3%, p <0.001). Of SOM-IR, SP-IR and VIP-IR varicosities that lacked VAChT-immunoreactivity, only 1 ± 0.3%, 0 ± 0.3% and 0% contained α-synuclein-immunoreactivity, respectively. 71 ± 0.8% of VAChT-IR varicosities in myenteric ganglia of human colon were α-synuclein-IR. In guinea-pig rectal myenteric ganglia, α-synuclein- and VAChT-immunoreactivity co-existed in 15 ± 1.4% of biotinamide-labeled extrinsic varicosities; only 1 ± .3% of biotinamide-labeled extrinsic varicosities contained α-synuclein-immunoreactivity without VAChT-immunoreactivity. α-Synuclein expression in axons to the distal gut correlates closely with expression of the cholinergic marker, VAChT. This is the first report of cell-selective α-synuclein expression in the nervous system. Our results suggest cholinergic neurons in the gut may be vulnerable in Parkinson's disease. J. Comp. Neurol., 2012. © 2012 Wiley Periodicals, Inc.