The release of motilin from an isolated preparation of pig duodenum has been studied. Three different types of stimuli were applied: electrical nerve stimulation, intraarterially administered peptides, and instillation of test solutions into the lumen of the duodenum. Furthermore, extracts of 20 different regions of the pig digestive system have been analyzed for motilin content. Analysis of the extracts only detected significant presence of motilin in the pig duodenum and jejunum (79 ± 15 and 60 ± 19 pmol/g). The stimulation experiments showed: (1) a significant noncholinergic depression of motilin release during electrical stimulation of the vagus nerve (nadir at 74 ± 5% of baseline level; (2) a significant elevation of motilin release in response to intraarterially administered vasoactive intestinal peptide (VIP) (peak at 330 ± 35% of baseline level), and (3) a significantly elevated motilin release in response to instillation of autologuous bile (peak at 170 ± 16% of baseline level) and hydrochloric acid (peak at 196 ± 42% of baseline level) into the duodenal lumen. In conclusion, luminal acidification and bile are important factors in stimulation of motilin release, whereas the vagally stimulated VIP release was insufficient to overcome the general inhibitory effect of vagus stimulation.