Paroxetine was shown to be a potent (K _i =1.1 nM) and specific inhibitor of [³H]-5-hydroxytryptamine (5-HT) uptake into rat cortical and hypothalamic synaptosomes in vitro. Lineweaver-Burk kinetic analysis determined that this inhibition was competitive in nature, implying a direct interaction with the 5-HT uptake transporter complex. Oral administration of paroxetine produced a dose-related inhibition of [³H]-5-HT uptake (ED ₅₀=1.9 mg/kg) into rat hypothalamic synaptosomes ex vivo with little effect on [³H]-l-noradrenaline (NA) uptake (ED ₅₀>30 mg/kg). This selectivity for 5-HT uptake was maintained after oral dosing for 14 days. Paroxetine (ED ₅₀ 1–3 mg/kg PO) prevented the 5-HT depleting effect of p-chloroamphetamine (PCA) in rat brain, demonstrating 5-HT uptake blockade in vivo. Radioligand binding techniques in rat brain in vitro showed that paroxetine has little affinity for ₁, ₂ or adrenoceptors, dopamine (D₂), 5-HT₁, 5-HT₂ or histamine (H₁) receptors at concentrations below 1000 nM. Paroxetine demonstrated weak affinity for muscarinic receptors (K _i =89 nM) but was at least 15 fold weaker than amitriptyline (K _i =5.1 nM). Paroxetine, therefore, provides a useful pharmacological tool for investigating 5-HT systems and furthermore should be an antidepressant with reduced tricyclic-like side-effects.