We examined the effect of morphine on the release of acetylcholine (ACh) in the striatum of freely moving rats using the in vivo microdialysis method. The basal level of ACh was 3.01 +/- 0.51 pmol/30 microliters/15 min in the presence of neostigmine (10 microM). Tetrodotoxin (1 microM), a selective blocker of voltage-dependent Na+ channels, rapidly decreased the release of ACh in the striatal perfusates. Morphine at a dose of 10 mg/kg (i.p.) caused a reduction of ACh release in the striatum at 90-150 min. However, a lower dose of morphine (5 mg/kg, i.p.) did not affect ACh release in the striatum. The reduction following intraperitoneal administration of morphine was abolished by naloxone (1.0 mg/kg). After microinjection of the neurotoxin 6-hydroxydopamine (6 micrograms/3 microliters, 7 days before) in the substantia nigra, the morphine (10 mg/kg)-induced decrease of ACh was attenuated, and a similar result occurred following reserpine (2 mg/kg, i.p.) 24 h before combined with alpha-methyl-p-tyrosine (300 mg/kg, i.p.) 2.5 h before. These findings indicate that morphine exerts an inhibitory influence on striatal ACh release in freely moving rats and that this inhibitory effect is mediated by the nigro-striatal dopaminergic system.