Mammalian lungs have been shown to store and to inactivate serotonin by an active process involving uptake and metabolism. Serotonin has direct action on lung including constrictor effects of pulmonary vascular and tracheobronchial smooth muscle suggesting the presence of serotonin receptors in lung. We have identified several serotonin binding receptors in rat lung. Two separate binding sites are present in a purified mitochondrial fraction. Saturation analysis of (3H)-serotonin binding to outer mitochondrial membranes exhibits temperature-dependent association kinetics and demonstrates a single, high affinity, high capacity binding (dissociation constant = 8.3 +/- 1.2 nM, maximum binding capacity = 0.819 +/- 0.046 pmol/mg protein). The dissociation constant of inner mitochondrial membrane demonstrates a low affinity, low capacity site (dissociation constant = 25.2 +/- 2.2 nM, maximum binding capacity = 0.453 +/- 0.037 pmol/mg protein). The purified microsomal fraction of lung exhibits a moderate affinity, high capacity binding site for (3H)-serotonin (dissociation constant = 14.8 +/- 1.6 nM, maximum binding capacity = 0.760 +/- 0.03 pmol/mg protein). In addition to the lung being the major site for its inactivation, the presence of several specific serotonin receptors may be related to some of the known serotonin actions in lung and may suggest other unknown actions of this amine.