Loop diuretics are widely used to inhibit the Na⁺-K⁺-2Cl^– cotransporter (Haas & Forbush, 2000). However, Venglarik (1997) demonstrated that loop-diuretics inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel. To understand better how loop diuretics inhibit CFTR, we studied furosemide, bumetanide, and two other agents xipamide and piretanide, which are structurally related to furosemide and bumetanide, respectively. We recorded CFTR Cl^– currents in inside-out membrane patches excised from C127 cells expressing wild-type human CFTR. The pipette (external) solution contained 10 mM Cl^– and the bath (internal) solution contained 147 mM Cl^–, 0.3 mM ATP and 75 nM PKA at 37°C; voltage was –50 mV. Data are means ± SEM of n observations and statistical analyses were performed using Student’s paired t test. When added to the internal solution, loop diuretics caused a reversible, concentration-dependent decrease in CFTR Cl^– current. For all agents tested, the concentration-response relationship was well fitted by the Hill equation with Hill coefficients of ~1. The rank order of potency for CFTR inhibition was xipamide (K_i = 45 ± 4 μM) ≥ bumetanide (K_i = 56 ± 11 μM) = piretanide (K_i = 58 ± 18 μM) ≥ furosemide (K_i = 71 ± 15 μM) (n = 5 for all agents). To investigate further channel block, we used noise analysis. In the absence of furosemide, power density spectra of CFTR Cl^– currents were best fitted with two Lorentzian components with corner frequencies f_c1 and f_c2 of 1.17 ± 0.6 and 81 ± 10 Hz (n = 4), whereas in the presence of furosemide (100 μM), power density spectra were best fitted with three Lorentzian components with f_c1, f_c2 and f_c3 of 2.64 ± 1.38, 63 ± 25 and 312 ± 107 Hz (n = 4), respectively, suggesting that f_c3 corresponds to the rapid binding and dissociation of furosemide to and from individual CFTR Cl^– channels. Consistent with this idea, furosemide (100 μM) caused a flickery block of CFTR decreasing both open probability (P_o: control, 0.41 ± 0.03; furosemide (100 μM), 0.17 ± 0.02; n = 6; p < 0.01) and single-channel current amplitude (i: control, –0.76 ± 0.01 pA; furosemide (100 μM), –0.63 ± 0.03 pA; n = 6; p < 0.01). Thus, our data demonstrate that loop diuretics inhibit CFTR, their potency approaches that of the widely used CFTR blocker glibenclamide and that furosemide acts an open channel blocker of CFTR.