Neurons that release serotonin as a neurotransmitter project to most regions of the central and peripheral nervous system and mediate diverse neural functions. The physiological effects of serotonin are initiated by the activation of multiple, distinct receptor subtypes. Cloning in RNA expression vectors was combined with a sensitive electrophysiological assay in Xenopus oocytes in order to isolate a functional cDNA clone encoding the 5HTlc serotonin receptor. Injection of RNA transcribed in vitro from this clone into Xenopus oocytes elicits serotonin sensitivity. Mouse fibroblasts transformed with this clone bind serotonin agonists and antagonists and exhibit an increase in intracellular Ca2+ concentrations in response to serotonin. The sequence of the 5HTlc receptor reveals that it belongs to the family of G protein-coupled receptors, which are thought to traverse the cytoplasmic membrane seven times. Moreover, in situ hybridization and RNA blot analysis indicate that the 5HTlc receptor is expressed in neurons in many regions of the central nervous system and suggest that this subclass of receptor may mediate many of the central actions of serotonin.