To explore whether Cl⁣ channel blockers interact with the ATP-sensitive K⁢ (KATP) channel, I have examined the effect of two common Cl⁣ channel blockers on the KATP channel activity in isolated rat ventricular myocytes using patch clamp techniques. In inside-out patches, 4,4'-diisothio-cyanatostilbene- 2,2'-disulfonic acid (DIDS) and niflumic acid applied to bath solution inhibited the KATP channel activity in a concentration-dependent manner with IC50 of 0.24 and 927μM, respectively. The inhibitory action of DIDS was irreversible whereas that of niflumic acid was reversible. Furthermore, DIDS-induced block was not recovered despite exposure to ATP (1 mM). In cell-attached and inside-out patches, DIDS blocked the pinacidil- or 2,4-dinitrophenol (DNP)-induced KATP channel openings. In contrast, niflumic acid did not block the pinacidil-induced KATP channel openings in inside-out patches, but inhibited it in cell-attached patches. DIDS and niflumic acid produced additional block in the presence of ATP and did not affect current-voltage relationship and channel kinetics. All these results indicate that DIDS among Cl⁣ channel blockers specifically blocks the cardiac KATP channel.