It has been shown that analgesics such as morphine, lidocaine and clonidine increase the release of spinal acetylcholine. Acetylcholine may therefore play an important role in the regulation of spinal pain threshold. Since behavioral as well as in vitro studies have shown a clear involvement of GABA (gamma-amino butyric acid) receptors in the regulation of spinal nociceptive mechanisms, the present study focused on the role of GABA receptors for spinal acetylcholine release control. GABA receptor agonists and antagonists were infused via a spinal microdialysis probe and acetylcholine release was measured. The GABA(A) receptor agonist muscimol decreased acetylcholine release and the antagonist bicuculline increased acetylcholine release. The GABA(B) receptor agonist baclofen decreased acetylcholine release whereas the antagonist saclofen did not change acetylcholine release. The results suggest that both GABA receptor subtypes have an inhibitory role on spinal dorsal horn acetylcholine release and that the GABA(A) receptors are tonically regulating acetylcholine release.