The cytochemical localization of acetylcholinesterase (AChE) at calyciform synapses of the chick ciliary ganglion during embryonic development has been investigated. AChE activity is present at the surface membrane of newly formed calyciform synapses and closely follows the progressive enlargement of the synaptic area. The occurrence of a retrograde iris-dependent influence on ganglionic AChE is considered. AChE seems to be a suitable marker for synaptic maturation.