It has been suggested that 5-hydroxytryptamine3 (5-HT3) receptors are involved in the control of phase III contractions in the stomach. We examined the effect of depletion of endogenous 5-HT by p-chlorophenylalanine (pCPA) on spontaneously and motilin-induced phase III contractions in conscious dogs, and the effect of 5,6-dihydroxytryptamine (5,6-DHT) in an isolated perfused dog stomach. Three-day treatment with pCPA significantly reduced plasma 5-HT concentration and 5-HT content in the stomach, and strongly suppressed the spontaneous and motilin-induced phase III contractions in the stomach. When spontaneous phase III contractions recovered in the stomach after a 3-day treatment, exogenous motilin induced typical phase III-like contractions, and the 5-HT content in the muscle layer was recovered to the normal pretreatment level. In the perfused stomach, 5,6-DHT decreased 5-HT content in the muscle layer alone and abolished motilin-induced contractions. In conclusion, endogenous 5-HT, probably in 5-HT neurons, plays an important role in the control of interdigestive phase III activity by motilin in the stomach.