The early appearance and functional significance of synaptic acetylcholinesterase (AChE) were examined in embryonic chick spinal cord-muscle cocultures. Synapses were identified by focal extracellular recording and divided into three categories based on their mean time constant of synaptic current decay ( ̄gtsyn): fast (1.0 msec < ̄gtsyn < 1.8 msec), intermediate (1.8 msec < ̄gtsyn < 2.6 msec), and slow ( ̄gtsyn > 2.6 msec). Patches of AChE histochemical reaction product were evident at identified synapses within 24 hr of coculture. After 4 days, 74% of the fast synapses stained for AChE, whereas none of the slow synapses was stained. Thus, it is likely that AChE serves to limit the duration of ACh action at newly formed nerve-muscle synapses, just as it does at the adult neuromuscular junction. This conclusion is supported by the fact that methanesulfonyl fluoride, an AChE inhibitor, prolonged synaptic currents at fast, but not at slow, synapses. Moreover, the mean channel open time of activated ACh receptors estimated by fluctuation analysis was the same at fast and slow synapses. © 1979.