Gut endocrine cells are generally thought to have distinct localization and secretory products. Recent studies suggested that the cells are highly related and have potential to express more than one hormone. We studied co-expression and co-secretion of gut hormones in separate segments of rat small intestine. We measured secretion of glucagon-like peptide-1 (GLP-1), peptideYY (PYY), neurotensin, glucose-dependent insulinotropic polypeptide (GIP), and cholecystokinin (CCK) from proximal and distal half of the small intestine, isolated from male rats and perfused ex vivo. Hormone secretion was stimulated by bombesin, the phosphodiesterase inhibitor IBMX, and peptones. Furthermore, tissue samples collected along the intestine were analyzed for expression, hormone content, and cell densities including colocalization. Most hormones responded to all three stimuli (but no GIP response to bombesin). GLP-1 secretion was similar from proximal and distal intestine, whereas PYY was only secreted from the distal half. CCK and GIP were mainly secreted proximally, whereas neurotensin was equally secreted from both parts. Cell densities, hormone concentrations and expression patterns were generally parallel, with increasing values distally for GLP-1 and PYY, an exclusively proximal pattern for CCK, even distribution for neurotensin and GIP except for the most distal segments. PYY nearly always co-localized with GLP-1. About 20% of GLP-1 cells co-localized with CCK and neurotensin, whereas GLP-1/GIP co-localization was rare. Our findings indicate that two L-cell types exist, a proximal one secreting GLP-1 (and possibly CCK and neurotensin), and a distal one secreting GLP-1 and PYY. GIP seems to be secreted from cells not co-secreting other peptides.