Patch clamp recordings were obtained from isolated cochlear efferent nerve terminals. The effect of aminoglycoside antibiotics on single maxi-K+ channels was determined. At positive voltages (cytosol with respect to extracellular side), neomycin, streptomycin, and kanamycin significantly reduced the single channel current amplitude of the maxi-K+ channel from the cytosolic side. The IC50 for neomycin was 9.10(-4) M from the cytosolic side and >> 10(-3) M from the extracellular side. Streptomycin and kanamycin were less potent. No significant difference in inhibition of the single channel current amplitude by 2.5.10(-4) M cytosolic neomycin was observed between 7.10(-4) M and 10(-6) M free cytosolic Ca2+. Neomycin had no significant effect on the open probability of the maxi-K+ channel either from the cytosolic or from the extracellular side. These findings demonstrate that the maxi-K+ channel in cochlear efferent nerve terminals can be a site of action for aminoglycoside antibiotics.