We measured cholinergic markers and acetylcholinesterase (AChE) molecular forms after glutamate receptor stimulation of superfused slices of mouse spinal cord at different developmental ages. AChE globular forms were secreted in a dose-dependent fashion. A period of selective sensitivity to excitotoxic agents was detected by increased acetylcholine (ACh) release and AChE secretion (sAChE) at postnatal day 14. Strychnine-resistant glycine stimulation potentiated glutamate-induced AChE release, suggesting N-methyl-D-aspartate (NMDA) receptor involvement.