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The present study used a binding assay to identify novel target biomolecules of l-menthol ([-]-menthol) that promote mouse ambulation. Among 88 different ligands to specific biomolecules examined, 0.1 mM l-menthol inhibited the binding of 13 ligands with relatively high inhibition rates. The assays showed that l-menthol acts on calcium channels, sodium channels, γ-aminobutyric acid type A (GABAA) receptor, GABA transporter, dopamine transporter, dopamine D4 receptor, adenosine A2a receptor, α2A-adrenergic receptor, histamine H2 receptor,