Rats were injected intracerebroventricularly with 5 microCi of [methyl-3H]choline. The time course of decline of the radiolabeled acetylcholine (ACh) formed was estimated in the ipsilateral cerebral cortex and striatum. The [3H]ACh levels declined biphasically from the cerebral tissue. The initial decline proceeded rapidly, after which labeled ACh declined more slowly. Scopolamine (1 mg/kg, i.v.) caused a significant increase in the rate of [3H]ACh disappearance, which can be interpreted as an enhancement of ACh release. By contrast, oxotremorine (0.8 mg/kg, i.v.) markedly reduced the [3H]ACh disappearance. The results show that drug-induced changes in cholinergic neuronal activities can be estimated from the disappearance of radioactive ACh after labeling the endogenous transmitter through intracerebroventricular administration of labeled choline.