Several lines of evidence have suggested that acetylcholine is a possible neurotransmitter/neuromodulator involved in vestibular compensation. Further, the central vestibular system, oculo- and spino-motor neurons and peripheral vestibular efferents contain abundant cholinergic neurons. However, details of cholinergic effective sites during vestibular compensation remain to be clarified. In the present study, we selectively damaged rat vestibulo-floccular and vestibulo-uvulonodular cholinergic mossy fibers using ethylcholine mustard aziridinium ions. In these treated animals, unilateral labyrinthectomy caused more severe vestibulo-ocular deficits especially during the initial stage.From these findings we suggest that vestibulo-floccular and vestibulo-uvulonodular cholinergic mossy fibers contribute to the restoration of a balance between intervestibular nuclear activities for the induction of vestibular compensation during the initial stage.