A mouse brain cDNA encoding the high-affinity serotonin transporter (SERT) has been identified and characterized. The mouse transporter sequence (mSERT) encodes a protein of 630 amino acids which contains twelve potential transmembrane domains (TMDs), N-linked glycosylation and kinase-mediated phosphorylation sites, and high levels of homology with rat and human SERTs. Heterologous expression of mSERT in COS-I cells resulted in a [3H]serotonin transport activity characterized by kinetic saturability (Km = 403 +/- 42 nM. Vmax = 1.02 +/- 0.10 pmol/mg/min), Na1 and Cl- dependences (5HT:Na+:Cl- coupling ratio of 1:1:1), and sensitivity to known inhibitors of serotonin transport (including antidepressant and psychostimulant agents). Northern analysis using mSERT cDNA as probe revealed a single 3.4 kb mRNA species expressed in mouse lung, midbrain and brainstem regions, and absent from heart and liver. In situ hybridization studies further established the specific localization of mSERT gene expression to the raphe nuclei of the mouse midbrain. The identified mSERT cDNA sequence provides a new tool for the evaluation of serotonin transport pharmacology in heterologous expression systems and provides an opportunity for the evaluation of mSERT gene expression in a well-characterized model of mammalian development.