Abstract The responses of the medial vestibular nucleus (MVN) neurons to lateral tilt and the neurotransmitters mediating otolith information to MVN neurons were investigated using rats. A computer-operated goniometer was tilted 20° clockwise and counterclockwise at an angular speed of 5°/s and paused in the inclined positions for 10 s to record neuronal responses in the static phase. The 185 MVN neurons recorded were classified into eight types according to their responses to tilt (α, β, γ, δ, ε, ζ, η and θ). A majority showed increased firing in response to ipsilateral tilting and decreased firing in response to contralateral tilting (α type: 31.4%) or exhibited the reverse pattern (β type: 36.8%). Further, other groups of neurons increased (γ type) or decreased (δ type) firing rates to either side tilting and increased (ε and ζ type) or decreased (η and θ type) firing only on one side. Atropine or l-glutamic acid diethyl ester hydrochloride (GDEE) applied microiontophoretically antagonized tilt-induced firing of α type neurons in 58.8% or 60.0%, respectively, and of β type neurons in 66.7% or 58.3%, respectively. When the effects of atropine and GDEE were examined in the same neurons, antagonizing effects of both drugs on tilt-induced firing were obtained in 28.6% and 40.0% of α and β type neurons, respectively. These results suggest that both acetylcholine and glutamate act as neurotransmitters in the transmission of otolith information to most MVN neurons.