The effect of botulinum toxin type A (BTX-A) on rat pyloric myoelectrical activity in vivo and the content and distribution of substance P (SP) in pylorus were investigated, respectively, with electromyography, radioimmunoassay, and immunohistochemistry. A pair of electrodes for recording pyloric myoelectrical activity and a guide cannula for drug injection were implanted into the pylorus. The changes of pyloric myoelectrical activity were recorded followed vehicle, 10, 20, and 40 U/kg body mass of BTX-A injection. Pyloric tissues were dissected for radioimmunoassay and immunohistochemistry after recording. The 3 dosages of BTX-A injections caused the reduction of slow wave of pyloric myoelectrical activity in amplitude but not in frequency and the diminishment of spike activity in amplitude and spike burst. The inhibitory effect of 20 U/kg BTX-A was significantly different from that of 10 U/kg (p<0.05), but not from the effect of 40 U/kg administration (p>0.05). After BTX-A intrasphincteric injection, SP content was reduced in the pylorus, and cell number of SP-immunoreactivity was decreased more in myenteric nerve plexus of circular muscle and in mucosa of pylori. In conclusion, BTX-A inhibits pyloric myoelectrical slow activity in amplitude and spike activity and weakens pyloric smooth muscle contractility depending on threshold of dose or concentration. BTX-A-induced inhibition of pyloric myoelectrical activity implies a mechanism of inhibiting SP release from the autonomic and enteric nervous terminals in the pylorus.