1. Simultaneous intracellular recordings were made from longitudinal muscle (LM) and circular muscle (CM) cells of guinea-pig distal colon during the peristaltic reflex. 2. Spontaneous rhythmical depolarizations with superimposed action potentials (mean amplitude: 19 ± 2 mV) were regularly recorded from the LM (mean interval: 7 ± 1 s). In contrast, in the CM layer, spontaneous action potentials occurred with an irregular frequency. Although spontaneous action potentials in LM were rarely correlated in time with those in CM, spontaneous inhibitory junction potentials (sIJPs) were found to occur synchronously in both muscles (5 out of 27 animals; 19%). 3. Graded inflation of an intra-luminal balloon or mucosal stimulation oral to the recording electrodes elicited gradeable compound IJPs synchronously in both LM (mean amplitude: 6 ± 1 mV) and CM (mean amplitude: 9 ± 1 mV) (descending inhibitory reflex). Evoked IJPs were often followed by action potentials in both muscle layers. 4. Mucosal stimuli applied anal to the recording electrodes elicited compound excitatory junction potentials (EJPs) synchronously in both muscles layers that were often associated with the generation of action potentials. In the LM, evoked EJP amplitudes ranged from 3 mV (subthreshold) to 31 mV (including the action potential) and in the CM from 4 mV (subthreshold) to 44 mV (including the action potential). 5. Apamin (500 nM) reduced the evoked IJP in the CM by 55% (from 11 ± 2 to 5 ± 1 mV), but caused no significant reduction in the LM layer (from 8 ± 1 to 6 ± 1 mV). Apamin-resistant IJPs in both muscle layers were likely to be due to nitric oxide, since they were abolished by L-NA (100 μM). 6. Atropine (1 μM) abolished the ascending excitatory reflex in both muscles. 7. Injection of neurobiotin into the LM and CM confirmed that simultaneous intracellular recordings were made from different muscle layers. 8. In conclusion, during the peristaltic reflex, the LM and CM layers receive synchronous inhibitory neuromuscular inputs during descending inhibition and synchronous excitatory neuromuscular inputs during ascending excitation. No evidence was found to support reciprocal innervation.