The possible presence of a luminal release of serotonin (5-HT) from gut enterochromaffin cells (EC) of the rat, was studied after the injection of the tritiated 5-HT precursor 5-hydroxytryptophan (3H]-5-HTP by electron microscopic autoradiography. The uptake of 5-HTP into gut epithelial cells was also studied by fluorescence histochemistry according to the Hillarp-Falck technique at the same post-injection interval as in the autoradiography experiments. 3 h after the injections of 5-HTP (100 mg/kg i.v.) the fluorescence intensity of EC was increased and numerous, probably enteroendocrine, cells had an increased yellow tryptamine induced fluorescence due to an uptake of 5-HTP and probably decarboxylation to 5-HT. However, the labelled precursor [3H]-5-HTP was taken up not only into granules of enteroendocrine cells but also incorporated into the cytoplasm and nucleus of nonendocrine cells when studied by autoradiography. After 10 min of efferent electrical stimulation of the vagal nerve much of the label was found in the gut lumen suggesting a release of the amine. The hypothesis of a luminal release of 5-HT was further corroborated in starved cats, where considerable amounts of 5-HT were detected by fluorimetric assays in the lumen of isolated jejunal loops under resting conditions. The experiments demonstrate that :(i) 5-HTP is taken up not only into typical EC but also into other enteroendocrine cells, and most probably decarboxylated to 5-HT. (ii) Also intestinocytes take up [3H]-5-HTP and incorporate the amino acid into peptides to a certain extent. (iii) Following vagal nerve stimulation labelled material, probably 5-HT, is secreted into the gut lumen. (iv) 5-HT normally occurs in the gut lumen.