Transverse tubular (TT) vesicles from rabbit skeletal muscle were incorporated into planar lipid bilayers to characterize the chloride channel. The single channel conductance of the channel was 40 pS in choline-Cl solution (cis, 300 mM/100 mM, trans). The gating rate of the channel does not depend on membrane voltage. The channel was blocked by stilbene derivatives (DIDS and SITS), which are known as inhibitors of voltage-dependent Cl- channels of the Torpedo electric organ, from both sides of the membrane. An inhibitor of voltage-dependent Cl- channels of skeletal muscles, 9-anthracene carboxylic acid (9-AC) inhibited the channel from the cis side of the membranes, which corresponded to the cytoplasmic space. Ethacrynic acid (EA), which is reported to inhibit Cl- conductance of the kidney and trachea, decreased the open probability of the TT Cl- channel concentration dependently. Indanyloxyacetic acid (IAA), which is also reported to be an inhibitor of kidney and trachea Cl- channels, decreased the single channel current without affecting open probability of the TT Cl- channel.