Parkinson’s disease is a neurodegenerative disorder characterized by Lewy bodies and neurites, which are composed mainly of the presynaptic protein, α-synuclein. Frequently, Lewy bodies and neurites are identified in the gut of Parkinson’s disease patients and may underlie associated gastrointestinal dysfunction. We recently reported selective expression of α-synuclein in the axons of cholinergic neurons in the guinea-pig and human distal gut; however, it is not clear if α-synuclein expression varies along the gut, nor how closely expression is associated with other synaptic proteins. We used multiple-labeling immunohistochemistry to quantify which neurons in the guinea-pig ileum expressed α-synuclein, cysteine string protein-α (CSPα), synaptophysin, synaptotagmin-1, or synaptobrevin-2 in their axons. Of the 10 neurochemically-defined axonal populations, a significantly greater proportion of vesicular acetylcholine transporter-immunoreactive (VAChT-IR) varicosities (80 ± 1.7%, n=4, P < 0.001) contained α-synuclein-immunoreactivity, and a significantly greater proportion of α-synuclein-IR axons also contained VAChT-immunoreactivity (78 ± 1.3%, n=4), compared to any of the other 9 populations (P < 0.001 in all cases). Of synaptophysin-, synaptotagmin-1-, synaptobrevin-2-, and CSPα-IR varicosities, 98 ± 0.7%, 96 ± 0.7%, 88 ± 1.6% and 85 ± 2.9% (n=4) contained α-synuclein-immunoreactivity, respectively. Of α-synuclein-IR varicosities, 96 ± 0.9%, 99 ± 0.6%, 83 ± 1.9% and 87 ± 2.3% (n=4) contained synaptophysin-, synaptotagmin-1-, synaptobrevin-2-, and CSPα-immunoreactivity, respectively. We report a close association between the expression of α-synuclein and other synaptic proteins in cholinergic axons in the guinea-pig ileum. Selective expression of α-synuclein may relate to the neurotransmitter system utilized and predispose cholinergic enteric neurons to neurodegeneration in Parkinson’s disease.