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 (GABA
A
) receptor, GABA transporter, dopamine transporter, dopamine D4 receptor, adenosine A2a receptor, α2A-adrenergic receptor, histamine H2 receptor, bombesin receptor, angiotensin AT1 receptor, vasopressin V2 receptor, and leukotriene B4 receptor over a similar concentration range. The inhibition constant (K
i
) for
l
-menthol inhibition of binding of [
3
H]-WIN35,428 to the human recombinant dopamine transporter was 6.15 × 10
−4
mol/L. The K
i
for
l
-menthol inhibition of binding of [
3
H]-ethynylbicycloorthobenzoate (EBOB), a ligand of GABA
A
receptor picrotoxin site, was 2.88 × 10
−4
mol/L. These results should aid future research by providing clues for investigating the mechanisms underlying
l
-menthol activities, including the ambulation-promoting effect. The present results suggest that the dopamine transporter, adenosine A2a receptor, dopamine D4 receptor, α2A-adrenergic receptor, and GABA
A
receptor are promising candidate molecules that are involved in the mechanisms underlying the psychostimulant-like effect of
l
-menthol.