Aim Gastrointestinal smooth muscle relaxation is accomplished by the neural corelease of ATP or a related purine and nitric oxide. Contractions are triggered by acetylcholine and tachykinins. The aim of this work was to study whether regional differences in neurotransmission could partially explain the varied physiological roles of each colonic area. Methods We used electrophysiological and myography techniques to evaluate purinergic (L‐NNA 1 mm incubated tissue), nitrergic (MRS2500 0.3 μm incubated tissue) and cholinergic neurotransmission (L‐NNA 1 mm and MRS2500 0.3 μm incubated tissue) in the proximal, mid and distal colon of CD1 mice (n = 42). Results Purinergic electrophysiological responses elicited by single pulses (28 V) were greater in the distal (IJPfMAX = −35.3 ± 2.2 mV), followed by the mid (IJPfMAX = −30.6 ± 1.0 mV) and proximal (IJPfMAX = −11.7 ± 1.1 mV) colon. In contrast, nitrergic responses decreased from the proximal colon (IJPsMAX = −11.4 ± 1.1 mV) to the mid (IJPsMAX = −9.1 ± 0.4 mV), followed by the distal colon (IJPsMAX = −1.8 ± 0.3 mV). A similar rank of order was observed in neural mediated inhibitory mechanical responses including electrical field stimulation‐mediated responses and neural tone. ADPβs concentration–response curve was shifted to the left in the distal colon. In contrast, NaNP responses did not differ between regions. Cholinergic neurotransmission elicited contractions of a similar amplitude throughout the colon. Conclusion An inverse gradient of purinergic and nitrergic neurotransmission exists through the mouse colon. The proximal and mid colon have a predominant nitrergic neurotransmission probably due to the fact that their storage function requires sustained relaxations. The distal colon, in contrast, has mainly purinergic neurotransmission responsible for the phasic relaxations needed to propel dehydrated faeces.