Agomelatine (S20098) displayed pKi values of 6.4 and 6.2 at native (porcine) and cloned, human (h)5-hydroxytryptamine (5-HT)2C receptors, respectively. It also interacted with h5-HT2B receptors (6.6), whereas it showed low affinity at native (rat)/cloned, human 5-HT2A (<5.0/5.3) and 5-HT1A (<5.0/5.2) receptors, and negligible (<5.0) affinity for other 5-HT receptors. In antibody capture/scintillation proximity assays, agomelatine concentration dependently and competitively abolished h5-HT2C receptor-mediated activation of Gq/11 and Gi3 (pA2 values of 6.0 and 6.1). As measured by [3H]phosphatidylinositol depletion, agomelatine abolished activation of phospholipase C by h5-HT2C (pKB value of 6.1) and h5-HT2B (pKB value of 6.6) receptors. In vivo, it dose dependently blocked induction of penile erections by the 5-HT2C agonists (S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine (Ro60,0175) and 1-methyl-2-(5,8,8-trimethyl-8H-3-aza-cyclopenta[a]inden-3-yl) ethylamine (Ro60,0332). Furthermore, agomelatine dose dependently enhanced dialysis levels of dopamine in frontal cortex of freely moving rats, whereas they were unaffected in nucleus accumbens and striatum. Although the electrical activity of ventrotegmental dopaminergic neurons was unaffected agomelatine, it abolished their inhibition by Ro60,0175. Extracellular levels of noradrenaline in frontal cortex were also dose dependently enhanced by agomelatine in parallel with an acceleration in the firing rate of adrenergic cell bodies in the locus coeruleus. These increases in noradrenaline and dopamine levels were unaffected by the selective melatonin antagonist N-[2-(5-ethyl-benzo[b]thien-3-yl)ethyl] acetamide (S22153) and likely flect blockade of 5-HT2C receptors inhibitory to frontocortical dopaminergic and adrenergic pathways. Correspondingly, distinction to agomelatine, melatonin showed negligible activity 5-HT2C receptors and failed to modify the activity of adrenergic and dopaminergic pathways. In conclusion, in contrast to melatonin, agomelatine behaves as an antagonist at 5-HT2B and 5-HT2C receptors: blockade of the latter reinforces frontocortical adrenergic and dopaminergic transmission.