The effect of two substances (α-bungarotoxin, α-BGTX, a small protein, and the local anesthetic bupivacaine hydrochloride) with an assumed effect on outer hair cell (OHC) motility were analyzed after exposing the cochlea via the round window membrane. Electrophysiological measurements were performed with a very narrow frequency-specific gating ( ± 100 Hz) technique to determine auditory brainstem response (ABR) thresholds, including ABR-based frequency tuning curves. Exposure to α-BGTX gave a minor improvement in thresholds, interpreted as a facilitation of OHCs, i.e. releasing their efferent inhibitory control, whereas exposure to bupivacaine hydrochloride impaired ABR thresholds, possibly due to immobilization of OHC motility via the lateral cell membrane. Our results are consistent with the hypothesis that efferent influence on the cochlea may be linked with a modulation of the mechanical function of OHCs. We can now postulate that there is in vivo evidence that acetylcholine exerts its effect at the OHCs via an α-BGTX binding acetylcholine rece