Ca2(+)-activated K+ channels from rat brain synaptosomal membranes were incorporated into planar lipid bilayers, and the effects of aminoglycoside antibiotics on the single channel conductance (258 +/- 13 pS at 100 mM K+) were investigated. Aminoglycosides reduced the single channel conductance from the 'cis' (cytoplasmic) side in a dose- and voltage-dependent manner. Voltage dependence of the blockade indicated an interaction between positively charged amino residues of aminoglycoside antibiotics and a binding site located within the electric field of the ion-conducting pathway. The order of blocking potency was consistent with that of the number of amino residues of aminoglycosides (neomycin (6) greater than dibekacin (5) greater than ribostamycin (4) = kanamycin (4], while the electrical distance (z delta = 0.46-0.49) of the binding site kept almost constant for each drug. These z delta s were almost the same with those (0.46-0.51) of alkyl-diamine blockers with two amino residues (total net charge of +2) and approximately twice of those (0.25-0.26) of alkylmonoamine blockers (total net charge of +1). Assuming that amino residues of aminoglycosides and alkylamines shared the same binding site located at 25% voltage drop from the cytoplasmic surface of the channel, the site would have to be at least large enough to accommodate one diamino sugar residue of the aminoglycoside in order to simultaneously interact with two positively charged amino groups. Dose- and voltage-dependent blockade of the channel by gallamine, an extremely bulky trivalent organic cation, supported the picture that the channel has a wide mouth on the cytoplasmic side and its 'pore' region, where voltage drop occurs, may also be quite wide and nonselective, suddenly tapering to a constriction where most charged cations block the channel by 'occluding' the K(+)-conducting pathway.