In a randomized, placebo-controlled crossover design we studied the effect of gastric acidification on motilin-induced interdigestive antropyloroduodenal motility. Ten healthy volunteers participated in the study consisting of four experiments. Each experiment started after a spontaneous occurring phase III and consisted of intragastric infusion of either saline or acid (0.08 mol L−1 HCl) for 90 min and intravenous infusion of either saline or motilin (4 pmol kg−1 min−1) for 30 min. Antropyloroduodenal motility and pH were recorded continuously for 240 min. Reoccurrence of phase III was significantly (P < 0.05) earlier during intragastric saline-intravenous motilin infusion compared with control (intragastric saline-intravenous saline), 52 min (range 25–79) and 113 min (84–141) respectively. This effect was completely abolished during intragastric acid-intravenous motilin infusion, 112 min (82–142). The percentage of phase III of antral origin was significantly (P < 0.05) higher during intragastric saline-intravenous motilin infusion (90%) compared with control (30%). The mean area under the contraction (AUC) for phase II was significantly (P < 0.05) lower during intragastric saline-intravenous motilin infusion and intragastric acid-intravenous saline infusion compared with control. It is concluded that in humans intragastric acidification inhibits the effect of motilin on antroduodenal motility, decreases the AUC of antral phase II contractions and delays the occurrence of phase III of the migrating motor complex.