Previous studies revealed that cholinergic neurons possessing long axons are extremely sensitive to ethylcholine aziridinium ion (AF64A) administration [Neuropharmacology 31 (1992) 397]. In the present paper we examined the effect of AF64A on the cholinergic elements of the cingulum bundle. Seven days after AF64A administration choline acetyltransferase (ChAT)-immunoreactive fibers were extensively damaged on the dorsal part of cingulum bundle. These findings are the first reporting damage by AF64A to this brain region.