The pyloric sphincter (PS) controls gastric emptying and prevents the reflux of duodenal content into the stomach. Neuronal pathways and reflexes controlling the guinea-pig PS were physiologically investigated in isolated preparations. Simultaneous intracellular or extracellular and tension recordings from PS circular muscle with electrical and stretch stimulation were used. Electrical stimulation evoked an initial small contraction followed by a relaxation with a corresponding inhibitory junction potential (IJP) then a second large contraction with a corresponding excitatory junction potential (EJP). Hyoscine (1 μmol L–1) blocked the first contraction, and reduced the second contraction and EJP by 52.5% and 61%, respectively. These responses were further reduced by the NK2 antagonist, MEN10627 (1 μmol L–1), and the NK1 antagonist, SR140333 (1 μmol L–1). N-nitro-L-arginine (100 μmol L–1) and apamin (0.5 μmol L–1) blocked the relaxation and the IJP. Duodenal electrical stimulation evoked an EJP, whereas antral stimulation evoked an IJP followed by a small EJP. All were blocked by hexamethonium (100 μmol L–1). Duodenal stretch evoked tetrodotoxin-sensitive reflex contractions and membrane depolarization with action potentials in the PS. Thus, PS enteric motor neurones receive inputs from the duodenum and the stomach. There are stretch-sensitive ascending excitatory reflex pathways from the duodenum to the PS.