The acetylcholine transporter exhibits such low affinity and specificity for acetylcholine that it appeared possible it could fail to select against other neurotransmitters. Potential interactions of classical noncholinergic neurotransmitters with cholinergic synaptic vesicles purified from electric organ were studied. No active transport of [3H]serotonin, [3H]noradrenaline, or [3H]glutamate occurred. Serotonin, noradrenaline, and N-acetyl-aspartyl glutamate inhibited active transport of [3H]acetylcholine by the vesicles. Dopamine previously had been shown to inhibit transport. Glutamate and gamma-aminobutyric acid were shown here not to inhibit active transport of [3H]-acetylcholine. Noradrenaline was competitive with respect to [3H]acetylcholine in this effect. Serotonin, noradrenaline, and dopamine inhibited binding of [3H]vesamicol to the vesicles, and dopamine was a competitive inhibitor of the binding of this allosteric ligand of the acetylcholine transporter. The results indicate that the acetylcholine transporter does not transport any other classical neurotransmitter, but serotonin, noradrenaline, and dopamine bind to the acetylcholine site.