The objective of these experiments was to confirm the localization of neurotensin (NT) in gut endocrine cells of the canine small intestine using immunohistochemistry. In addition, the release of NT from the canine small intestine in response to selective perfusion of a fatty acid (oleate), triglyceride (Lipomul) or products of fat digestion into various segments of the small intestine was studied. In the immunohistochemical study, NT was found to be primarily localized in true endocrine cells of the ileal mucosa. In addition, NT was not found or only negligible numbers of cells were seen outside the lower small intestine. This observation supports previous results based on radioimmunoassay and immunohistochemistry studies. Based on these morphological findings, NT would be released by luminal secretagogues, of which fat appears to be the most potent. In the selective perfusion studies, perfusion of oleic acid into the jejunum of the chronic dog caused NT release, whereas perfusion of the ileum in which NT cells were most abundant was ineffective. This observation suggests that a neural or endocrine message is released to the ileal NT cell from the jejunum, causing NT release. This series of studies was carried out to elucidate the mechanism of NT release and to find the direct luminal stimulants of NT by using both chronic and acute experimental models. These studies suggest that NT is not significantly released under anesthesia and that undigested fat, like triglyceride, does not release NT in either the upper or lower small intestine. Furthermore, digested fat, like oleate or digestive juices in the lower small intestine, is not a direct stimulant of NT release.