Lumbar splanchnic (LSN) and sacral pelvic (PN) nerves convey different mechanosensory information from the colon to the spinal cord. Here we determined whether these pathways also differ in their chemosensitivity and receptor expression. Using an in vitro mouse colon preparation, individual primary afferents were tested with selective P2X and transient receptor potential vanilloid receptor 1 (TRPV1) receptor ligands. Afferent cell bodies in thoracolumbar and lumbosacral dorsal root ganglia (DRG) were retrogradely labelled from the colon and analysed for P2X 3 -and TRPV1-like immunoreactivity (LI). Forty per cent of LSN afferents responded to α,β-methylene adenosine 5 -triphosphate (α,β-meATP; 1 mM), an effect that was concentration dependent and reversed by the P2X antagonist pyridoxyl5-phosphate 6-azophenyl-2 ,4 -disulphonic acid (PPADS) (100 µM). Significantly fewer PN afferents (7%) responded to α,β-meATP. Correspondingly, 36% of colonic thoracolumbar DRG neurones exhibited P2X 3 -LI compared with only 19% of colonic lumbosacral neurones. Capsaicin (3 µM) excited 61% of LSN afferents and 47% of PN afferents; 82% of thoracolumbar and 50% of lumbosacral colonic DRG neurones displayed TRPV1-LI. Mechanically insensitive afferents were recruited by α,β-meATP or capsaicin, and were almost exclusive to the LSN. Capsaicin-responsive LSN afferents displayed marked mechanical desensitization after responding to capsaicin, which did not occur in capsaicin-responsive PN afferents. Therefore, colonic LSN and PN pathways differ in their chemosensitivity to known noxious stimuli and their corresponding receptor expression. As these pathways relay information that may relate to symptoms in functional gastrointestinal disease, these results may have implications for the efficacy of therapies targeting receptor modulation.