Endocrine L-cells of the distal intestine synthesize both peptide YY (PYY) and proglucagon-derived peptides (PGDPs), whose release has been reported to be either parallel or selective. Here we compare the release mechanisms of PYY, glucagon-like peptide-1 (GLP-1), and oxyntomodulin-like immunoreactivity (OLI) in vivo. Anaesthetized rats were intraduodenally (ID) given either a mixed semi-liquid meal or oleic acid, or they received oleic acid or short chain fatty acids (SCFA) intracolonically (IC). The ID meal released the three peptides with a similar time-course (peak at 30 min); ID oleic acid produced a progressive release of PYY and OLI, while GLP-1 release was less. IC oleic acid or SCFA released smaller (but significant) amounts of PYY but no OLI or GLP-1. Hexamethonium inhibited most of the response to the ID meal and ID oleic acid, but did not change the PYY response to IC oleic acid. NG-nitro-l-arginine methyl ester (l-NAME, a nitric oxide synthase inhibitor) inhibited meal-induced PYY release and left OLI and GLP-1 unaffected. BW10 (a gastrin-releasing peptide antagonist) had no effect on the meal-induced release of either peptide. These results suggest a parallel initial release of PYY, OLI and GLP-1 after the ID meal, or oleic acid, by an indirect mechanism triggered in the proximal bowel, using nicotinic synapses, and involving nitric oxide release for PYY and an unknown mediator for PGDPs. For PYY there is a later phase of peptide release, probably induced by direct contact between nutrients and colonic L-cells.