The 5-HT levels in biopsies from the rat gastrointestinal tract were measured by a sensitive liquid chromatography method with electrochemical detection. The highest levels were found in the gastroduodenal and cecal regions. Sham-operated controls were compared with rats subjected to combined truncal vagotomy and pyloroplasty or pyloroplasty alone 5 weeks after surgery. Significantly decreased 5-HT levels were demonstrated in animals subjected to vagotomy + pyloroplasty or to pyloroplasty alone, compared with sham-operated controls. In the upper gastrointestinal tract of vagotomized animals significantly decreased 5-HT levels were found when compared with animals subjected to pyloroplasty alone. However, anesthesia and surgery (sham operation) per se markedly increased 5-HT levels of the gut even 5 weeks postoperatively, when compared with acutely killed rats. The underlying mechanism is still obscure. However, vagotomy largely prevented this increase. Therefore, the importance of correct controls is emphasized, since previous investigations claim to have demonstrated increased 5-HT levels in vagotomized animals, when compared with nonoperated controls.