To test the hypothesis that the distension-induced esophageal peristaltic reflex involves a polysynaptic pathway, a triple-chamber organ bath was used to chemically isolate different regions of the opossum smooth muscle esophagus while leaving in continuity the intramural neuromuscular apparatus. Balloon distension in the oral chamber evoked membrane hyperpolarization in the aboral chamber, which was followed, on balloon deflation, by depolarization, spike burst, and circular smooth muscle contraction. This reflex was abolished by adding tetrodotoxin to any of the chambers. Addition of Ca(2+)-free 20 mM Mg2+ Krebs solution (to block synaptic transmission) to the intermediate chamber did not affect the descending peristaltic reflex. However, Ca(2+)-free 20 mM Mg2+ Krebs solution markedly attenuated the reflex when placed in either the oral or the aboral chamber. Furthermore, the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester, when placed in the aboral chamber, abolished the reflex. Other putative neurotransmitter antagonists were without effect when placed in any chamber. These studies suggest that the intramural neural pathway that mediates the descending peristaltic reflex in the opossum esophagus is not polysynaptic, but rather involves long descending neurons that depend on nitric oxide as a final mediator.