Activation of cholinergic receptors in the spinal cord increases the intraspinal release of acetylcholine (ACh) and produces potent analgesia. The mechanisms that regulate the release of spinal ACh are not fully known. In the present study, we investigated the role of nicotinic ACh receptors in the regulation of intraspinal ACh release. Using an in vivo intraspinal microdialysis technique, nicotine was administered alone and in combination with the nicotinic antagonists mecamylamine (50 microM), dihydro-beta-erythroidine (DbetaE) (500 microM) and methyllycaconitine (MLA) (40 nM). Administration of nicotine (1 microM-1 mM) produced a dose dependent increase of intraspinal ACh release, while 10 mM nicotine resulted in dramatic increase in ACh release followed by a decrease to baseline. Administration of mecamylamine or DbetaE also induced an increased ACh release while MLA caused a decreased release. Mecamylamine and DbetaE, but not MLA pretreatment attenuated the stimulatory effect of 100 microM nicotine on intraspinal ACh release. It is suggested that spinal ACh release is regulated by different nicotinic ACh receptors. These receptors may tonically regulate spinal ACh release either directly or indirectly via inhibitory interneurones. Some of these receptors may be desensitised by high nicotine concentrations leading to a reduction of ACh release.