Recently, we reported that 6R-L-erythro-tetrahydrobiopterin (6R-BH4), a natural cofactor for L-aromatic amino acid hydroxylases, enhances in vivo release of acetylcholine (ACh) in the rat hippocampus: the enhancement was abolished after depletion of brain catecholamines and 5-hydroxytryptamine (5-HT) by pretreatment with reserpine. In the present study, we have used in vivo brain microdialysis to clarify the neuronal mechanism involved in the enhancement of ACh release by 6R-BH4. After depletion of catecholamines by pretreatment of rats with alpha-methyl-p-tyrosine, 6R-BH4 added to the perfusion fluid still induced an increase in extracellular ACh levels monitored by microdialysis as an index of ACh release. In contrast, after depletion of 5-HT by pretreatment with p-chlorophenylalanine, most of the 6R-BH4-induced enhancement was eliminated. Exogenous 5-HT and dopamine (DA) but not noradrenaline added to the perfusion fluid stimulated ACh release with 5-HT being far more potent. Intraperitoneal administration of 5-hydroxytryptophan and L-DOPA also enhanced ACh release, presumably by their conversion to 5-HT and catecholamines, respectively. Administration of 6R-BH4 increased hippocampal 5-HT release, as indicated by increased extracellular levels of the major 5-HT metabolite, 5-hydroxyindoleacetic acid. These results suggest that 6R-BH4 stimulates ACh release in the hippocampus, mainly by augmenting release of 5-HT, a potent stimulator of ACh release, and partly by augmenting release of DA.