BACKGROUND AND OBJECTIVES: Unilateral vestibular loss can be induced by intratympanic gentamicin instillation. Despite accumulated reports on the morphologic changes after gentamicin treatment, there are limited reports regarding the effects of gentamicin ototoxicity on the vestibulo-ocular reflex (VOR), in especially unilateral vestibular deficit by local gentamicin instillation. Aim of this study is to provide the functional changes after local gentamicin application in guinea pigs. MATERIALS AND METHODS: Ten white guinea pigs (5 each for control and gentamicin treatment group) were used. Following surgical exposure of the left bulla, 0.9% saline solution or 40 mg/ml of gentamicin soaking gelfoam were applied on the round window. Horizontal vestibulo-ocular reflex (hVOR) was tested during earth vertical axis (EVA) sinusoidal harmonic acceleration rotation at 4 frequencies (0.04, 0.08, 0.16 and 0.32 Hz, 60 deg/sec of peak velocity). In one gentamicin treated animal, hVOR was tested during step velocity off-vertical axis rotation (OVAR) to clockwise and counterclockwise direction (30 degrees forward tilt from EVA with constant velocity of 100 deg/sec). Every test was repeated before treatment and at 2 days, 5 days and 7 days after treatment. RESULTS: The hVOR gain was significantly lowered after gentamicin treatment at all tested frequencies (p<0.05), while the gain does not change over time in control animals. Loss of bias component was evident and some decrease of modulation component was observed in counter-clockwise (lesion side) rotation after gentamicin treatment. CONCLUSION: This study provides characteristics of hVOR during EVA rotation and OVAR in unilateral vestibular deficit animal model by intrabullar gentamicin application.