In the developing vertebrate brain, acetylcholinesterase (AChE) expression coincides temporally with axon tract formation. Although AChE promotes neurite outgrowth in vitro, the role of this molecule in the development of axon tracts in vivo is unknown. To address this question, we examined the effects of the AChE inhibitor, BW284C51, on the formation of the early scaffold of axon tracts in the embryonic Xenopus brain. In exposed Xenopus brain preparations, axons elongate and establish a normal topography of axon tracts. However, when brains were exposed to BW284C51, the thickness of the major longitudinal axon tract, the tract of the post-optic commissure decreased in a dose-dependent manner. When BW284C51 was removed from the culture media axon tract development returned to normal within 5 h. These findings provide the first evidence for a non-classical role of AChE in the initial formation of axon tracts within the developing vertebrate brain.