Glycine is known as an inhibitory neurotransmitter in the spinal cord and forebrain but its precise role in the forebrain is largely overlooked. This investigation evaluated whether glycine alters acetylcholine, glutamate or dopamine release from striatal tissue using an in vitro approach. We observed that while glycine induced a robust 3H-acetylcholine release ( 3H-ACh) from superfused striatal tissue, it failed at releasing 3H-glutamate or 3H-dopamine. Glycine stimulated 3H-ACh release in a dose- and calcium-dependent manner (EC 50 = 69 μM). Tetrodotoxin (1 μM) inhibited about 75% of the release demonstrating a predominant dendritic and cell body location of glycine receptors. The prototypical glycine receptor antagonist strychnine at 10 μM completely abolished 3H-ACh release. To further characterize the role of striatal glycine receptors in 3H-ACh release we examined glycine effects after in vivo treatment with Haloperidol-decanoate (HD). Treatment for 30 days or more with HD decreased maximal glycine-stimulated release of 3H-ACh suggesting a non-competitive inhibition. After 30 days of washout release parameters did not return to vehicle-treated levels. The glutamate agonist NMDA also stimulated acetylcholine release but showed slightly different behavior in HD-treated striatal tissue. These effects could be attributed to changes in chloride transporters expressed in the giant striatal cholinergic cell as well as glycine receptor subunit composition and finally, GABA/glycine co-release in this tissue.