To study the mechanisms by which glutamate-elicited acetylcholinesterase release (GEAR) might play a part in the pathogenesis of excitotoxically triggered motor neurone disease, and to investigate the interaction of GEAR with spinal glycinergic mechanisms, we measured acetylcholinesterase (AChE) and cholinergic markers, after stimulating ventral horn slices and synaptosomes from the mouse spinal cord, with both glutamate- and glycine-receptor agonists. Glutamate (GLU), kainate and AMPA, as well as glycine (GLY) evoked dose-related, calcium-dependent liberation of soluble forms of AChE from both slices and synaptosomes. GLY-evoked AChE release showed remarkable age-related postnatal changes. In the immature slice of the ventral horn. GLY potentiated the GEAR response in the presence of strychnine, suggesting N-methyl-D-aspartate (NMDA) receptor involvement, and was also able to evoke a strychnine-sensitive AChE release in the absence of exogenous GLU. After the 28th postnatal day, nearly all the AChE secreted was released either after the activation of non-NMDA glutamate receptors or by strychnine-sensitive GLY-evoked AChE release mechanisms. Both GEAR and GLY-evoked AChE release might impair the negative feedback loop which modulates the overactivation of motor neurones, and cause prolonged extracellular rises of soluble AChE. These effects might augment the vulnerability of motor neurones to excitotoxic stress, promote fiber outgrowth, and eventually accelerate the metabolic exhaustion of lower motor neurones. It is possible that the mechanisms described are operative at the spinal cord of ALS/MND patients.