The uptake, metabolism and efflux of ¹⁴C-5-hydroxytryptamine (5-HT) were studied in isolated rat lungs, artificially ventilated and perfused via the pulmonary artery with Krebs' medium containing 5 mM glucose and 4.5% bovine serum albumin. Infusion of 0.11 to 17 µM ¹⁴C-5-HT resulted in concentration of radioactivity in the lungs. The uptake was a saturable process with the same K_m and V_max whether the main product found in the lungs was 5-hydroxyindoleacetic acid (control conditions) or 5-HT (treatment with iproniazid, an inhibitor of monoamine oxidase). This finding indicates that the rate-limiting step in the uptake of ¹⁴C-5-HT was its intracellular transport, not its subsequent metabolism. Once taken up, ¹⁴C-5-HT was located in the soluble fraction of lung homogenate. Cocaine, imipramine, chlorpromazine and cold markedly inhibited the pulmonary uptake of ¹⁴C-5-HT; anoxia, norepinephrine and tryptamine had only a partial inhibitor effect. Pretreatment with reserpine or perfusion with glucose-free medium had no effect on the uptake. Changes in the ionic composition of the medium were also studied: the absence of Na⁺ resulted in nearly complete inhibition of concentration of ¹⁴C-5-HT in the lungs, and lowered Na⁺, as well as increased K⁺, caused a decrease in the affinity of the transport system for ¹⁴C-5-HT. Ouabain at 10^-3 M had only a partial inhibitory effect. The efflux of ¹⁴C-5-HT from the lungs was accelerated by cocaine, imipramine or chlorpromazine, by the absence of extracellular Na⁺ and by low extracellular Na⁺. These results indicate that the mechanisms of uptake of 5-HT by perfused lungs, platelets and brain are similar and compatible with the model of Na⁺-dependent transport; however, the lungs, unlike the other cellular systems, do not store 5-HT, but metabolize it rapidly.