Effects of stilbene disulfonates on single KATPchannel currents were investigated in inside-out and outside-out membrane patches from guinea pig ventricular myocytes. All drugs tested, 4,4′-diisothiocyanatostilbene, 2,2′-disulfonic acid (DIDS), 4-acetamido0-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS), and 4,4′-diaminostilbene-2,2′-disulfonic acid (DADS), inhibited the KATPchannel when they were applied to the intracellular, but not extracellular side of the membrane patch. Inhibitory actions of DIDS and SITS were irreversible, whereas those induced by DNDS and DADS were reversible. KATPchannel inhibition was concentration dependent with an order of potency of DIDS>SITS ≈ DNDS>DADS; the Hill coefficient was close to unity for each drug. No change in channel conductance was observed during exposure to DIDS or DNDS; however, channel kinetics was altered. Distribution of the open time within bursts and that between bursts could be described by a single exponential relation in the absence and presence of DIDS or DNDS. The time constant of the open time within bursts was not altered, but that between bursts was decreased by DIDS (from 40.0±8.1 to 29.8±6.7 msec,P<0.05) and by DNDS (from 43.1±9.3 to 31.9±7.1 msec,P<0.05). Distributions of closed time within bursts were also fitted to a single exponential function both in the absence and presence of drugs, while those of the closed time between bursts were fitted to a single exponential function in the absence of drugs, but a double exponential function was required in the presence of drugs. The rates of onset and development of channel inhibition by DIDS and DNDS appeared to be concentration dependent; a longer time was required to reach a new steady-state of channel activity as drug concentration was decreased. Inhibition by DIDS or DNDS was regulated by intracellular pH; inhibition was greater during acidic conditions. For DIDS (0.1 mm), the open probability (Po) expressed as a fraction of the value before drug application was 42.9±8.3% at pH 7.4 and 8.2±6.6% at pH 6.5 (P<0.01); corresponding values for DNDS (1 mm) were 39.6±17.6 and 8.9 ±5.8%, respectively (P<0.01). From these data, we conclude that stilbene disulfonates block the KATPchannel by binding to their target site with one-to-one stoichiometry. Similar to glibenclamide, the binding of stilbene disulfonates may reflect interpolation in an “intermediate lipid compartment” between the cytosolic dru