Recently, we reported that intracerebroventricular administration of 6R-L-erythro-tetrahydrobiopterin (6R-BH4), a common natural cofactor for phenylalanine, tyrosine and tryptophan hydroxylases, enhances in vivo exocytotic release of acetylcholine (ACh) from the rat hippocampus, as monitored by brain microdialysis. In the present study, we developed a method for separation of ACh from 6R-BH4 on small columns of Sephadex G-10, which allowed us to determine by high pressure liquid chromatography (HPLC) the changes of ACh levels recovered in dialysates following addition of 6R-BH4 to the dialyzing fluid. Infusions of 6R-BH4 (final concentration, 0.25, 0.5 and 1 mM in the perfusing fluid) produced concentration-dependent increases in ACh levels in dialysates (1.5-, 3- and 8-fold of the basal levels, respectively), but was without effect on choline levels in dialysates. Infusion of 6S-L-erythro-5,6,7,8-tetrahydrobiopterin (6S-BH4), an unnatural diastereoisomer of 6R-BH4, or L-erythro-biopterin (biopterin), an oxidized form of 6R-BH4, did not affect the ACh levels in dialysates. The 6R-BH4-induced increase in ACh levels persisted after pretreatment with atropine, a muscarinic antagonist, but did not occur in rats pretreated with reserpine to deplete monoamines. These results show that 6R-BH4 stimulates exocytotic ACh release in the hippocampus through action on a local monoaminergic system in the hippocampus.