Several lines of evidence have suggested that acetylcholine is a possible neurotransmitter/neuromodulator involved in vestibular compensation. However, details of cholinergic effective sites during vestibular compensation remain unclear. In this study, we selectively damaged the rat vestibulo-floccular cholinergic mossy fibers using ethylcholine mustard aziridinium ion. In these animals, unilateral labyrinthectomy caused more severe vestibulo-ocular deficits, especially in the initial stage. These findings suggest that the vestibulo-floccular cholinergic mossy fibers serve to restore the balance between intervestibular nuclear activities in order to induce vestibular compensation in the initial stage.