In the present study, we have demonstrated that ACh is the predominant fast excitatory transmitter used by identified motor neurons innervating feeding muscles in Aplysia. A detailed study of ACh metabolism was then carried out in a well-characterized neuromuscular preparation, intrinsic muscle 5 (15). This neuromuscular system has a high-affinity uptake system for choline. The rate of uptake of choline was increased by motor neuron stimulation, and this increased uptake appears to be selectively targeted to motor neuron terminals. These properties appear similar to those observed in vertebrate neuromuscular preparations. However, we have made two observations that are surprising in light of our knowledge concerning the vertebrate neuromuscular junction where released ACh is rapidly hydrolyzed by acetylcholinesterase (AChE) to choline, which is then taken up by a high-affinity uptake system. This Aplysia neuromuscular system has limited endogenous AChE activity and contains a separate high-affinity uptake system for ACh itself that actually has a higher velocity than that for choline uptake. It is possible that the uptake system for ACh is involved in terminating the action of released transmitter in a manner similar to that previously described for noncholinergic transmitters. Using this preparation, we have demonstrated release of labeled ACh in response to intracellular stimulation of identified motor neurons. The release per spike appears to be highly plastic,increasing markedly with stimulation frequency. This preparation is amendable to study the regulation of release of peptide and conventional transmitters from the terminals of individual neurons.