The intraventricular administration of 6-hydroxydopamine (6-OHDA) depletes the striatum of dopamine (DA). When given to rat pups at an early age, the toxin also increases striatal serotonin (5-HT) content. In the accompanying report we observed that endogenous 5-HT, like DA, exerts an inhibitory influence on the release of acetylcholine (ACh) from striatal slices prepared from control animals and that the extent of this inhibition is related to the degree of serotonergic innervation of the region being examined. To determine whether this hyperinnervation was accompanied by an increase in serotonergic influence on ACh release, striatal slices were prepared from adult rats, preincubated with [3H]choline, superfused, and exposed to electrical field stimulation. The efflux of tritium into the superfusate was used as a measure of ACh release. In confirmation of previous reports, we observed that direct and indirect agonists of DA and 5-HT both reduced ACh overflow from control slices, whereas overflow was increased by antagonists of these amines. Slices prepared from rats given 6-OHDA-induced lesions as adults were responsive to each of these pharmacological manipulations, as well. In contrast, ACh overflow from slices prepared from animals lesioned with 6-OHDA as neonates was not modified by either dopaminergic or serotonergic drugs. These results suggest that the serotonergic hyperinnervation of striatum produced by neonatal 6-OHDA is accompanied by a loss of the inhibitory influence of endogenous 5-HT and DA on striatal ACh release and, thus, provide no evidence for a role for either transmitter in the behavioral sparing associated with such lesions.