The present investigation was designed to determine the pulmonary biosynthesis of 5-hydroxytryptamine (5-HT) from L-tryptophan. Artificially ventilated, isolated rabbit and rat lungs were perfused with a constituted medium. Tryptophan and its metabolites were detected by high pressure liquid chromatography using an electrochemical detector. 14C-tryptophan uptake by the rabbit lung was 5.6% and 3.9% in the rat lung after 1 hr of perfusion. The perfusate (100 ml) concentrations of 5-hydroxytryptophan and 5-hydroxytryptamine increased significantly (1.1 to 1.8 micrograms 5-HT and 4.4 to 6.5 micrograms 5-HTP) during rabbit lung perfusion. However, no metabolites were detected in the perfusate during rat lung perfusion. 5-Hydroxytryptamine and 5-hydroxyindoleacetic acid levels were greater in both rabbit and rat lungs when they were perfused with 0.4 mM tryptophan, compared to their levels in lungs perfused without tryptophan. The increase of 5-HT content in rat lung alone was statistically significant. 5-Hydroxytryptophan was not detected in the rabbit or rat lungs. These results suggest the presence of a mechanism for tryptophan metabolism in lung, similar to that in brain and gastrointestinal tract. However, since the rate of pulmonary metabolism of tryptophan is very low, pulmonary synthesis of 5-HT from tryptophan and its contribution to the circulating 5-HT pool is unlikely to be of significance.