The effect of various substrates on the rate of uptake of 5-hydroxytryptamine (serotonin) from the pulmonary circulation was studied with isolated guinea pig lungs. Lungs were ventilated and were perfused with an electrolyte solution in a recirculating system. Uptake of serotonin was calculated from the rate of disappearance of [14C]serotonin from the perfusate. Serotonin uptake was inhibited by approximately 40% when 5 mM 2-deoxyglucose was substituted for glucose in the pulmonary perfusate. The inhibition due to 2-deoxyglucose was reversed by addition to the perfusate of pyruvate, lactate, acetate, alanine, beta-hydroxybutyrate, palmitate, or glycerol. Fructose ans succinate were less efficient substrates while no significant effect was noted with L-glycerol 1-phosphate or L-phenylalanine. These results indicate that a wide variety of metabolizable substrates can be utilized by the pulmonary endothelium to support uptake of serotonin.