At early stages of embryonic development of the glossiphoniid leech, Theromyzon rude, a branch, termed MAC, of the axon of the segmentally iterated Retzius neuron extends into the anterior interganglionic connective nerve. At later stages, this branch disappears again in about 30% of the Retzius neurons in the standard midbody segments and in about 75% of the Retzius neurons in the two reproductive midbody segments. The frequency of disappearance of the MAC branch increases to about 85% in all Retzius neurons upon exposure of the embryos to culture media containing 1 mM acetylcholine (ACh) and 10 microM physostigmine during a sensitive period of axon outgrowth. This disappearance represents a retraction of the MAC branch to its point of origin, while other axon branches of the Retzius neuron remained unaffected. In later development, the retracted (medial) MAC branch was replaced by a new (lateral) branch termed LAC. The observations were made using confocal microscopy of fixed embryos stained with anti-5-HT antibody and confirmed by Lucifer yellow injection of individual Retzius neurons. The specific retraction of a single axon branch might be attributable to the local presence of extracellular matrix molecules in the ganglionic neuropil, which is contacted only by the MAC axon branch and could render this branch susceptible to growth-regulating signals. Since Retzius axon morphology in standard segments of ACh- treated embryos resembled that of reproductive segments in untreated embryos, it appears possible that ACh treatment may have simulated a process that contributes to the segmental differentiation of the Retzius neuron.