The expression and function of the serotonin transporter (SERT) is important in the regulation of mood and emotion. Determination of SERT alterations in physiological and pathological states is essential for understanding the role of SERT in mood regulation, and in the etiology and therapy of psychiatric disorders. Two SERT ligands, AFM ([(3)H]2-[2-(dimethylaminomethylphenylthio)]-5-fluoromethylphenylamine) and DASB ([(3)H]3-amino-4-[2-(dimethylaminomethylphenylthio)]benzonitrile), have recently been developed for positron emission tomography (PET) imaging. The aim of the present study was to determine the selectivity of these compounds for SERT. Autoradiography of AFM or DASB binding was compared in the brains of mice with genetically normal, diminished, or absent SERT. In addition, the pharmacodynamic profile of [(3)H]AFM was examined in the mouse brain. The distribution of [(3)H]AFM and [(3)H]DASB binding in the normal brains was consistent with that of previously studied serotonin reuptake inhibitors. Both ligands had negligible binding in the brain of SERT knockout mice, and binding was reduced approximately 50% in heterozygote SERT mice. The K(d) of [(3)H]AFM binding in the cortex and midbrain was 1.6 and 1.0 nM, respectively. Competition studies showed that [(3)H]AFM has very low affinity for norepinephrine and dopamine transporters as well as 5-HT receptors, including 5-HT(1A), 5-HT(1B), 5-HT(2A), and 5-HT(2C) receptors. In addition, fenfluramine showed a low capability to compete with [(3)H]AFM. The present results suggest that both AFM and DASB are highly selective SERT ligands potentially suitable for use in human PET studies of SERT.