Previous studies have shown that two indazole compounds, lonidamine [1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid] and its analogue AF2785 [(1-(2,4-dichlorobenzyl)-indazol-3-acrylic acid], suppress fertility in male rats. We also found that these compounds inhibit the cystic fibrosis transmembrane conductance regulator chloride (CFTR-Cl-) current in epididymal epithelial cells. To further investigate how lonidamine and AF2785 inhibit the current, we used a spectral analysis protocol to study whole-cell CFTR current variance. Application of Ionidamine or AF2785 to the extracellular membrane of rat epididymal epithelial cells introduced a new component to the whole-cell current variance. Spectral analysis of this variance suggested a block at a rate of 3.68 μmol-1/sec-1 and an off rate of 69.01 sec-1 for Ionidamine, and an on rate of 3.27 μmol-1/sec-1 and an off rate of 108 sec-1 for AF2785. Single CFTR-Cl- channel activity using excised inside-out membrane patches from rat epididymal epithelial cells revealed that addition of Ionidamine to the intracellular solution caused a flickery block (a reduction in channel-open time) at lower concentration (10 μM) without any effect on open channel probability or single-channel current amplitude. At higher concentrations (50 and 100 μM), Ionidamine showed a flickery block and a decrease in open-channel probability. The flickery block by Ionidamine was both voltage-dependent and concentration-dependent. These results suggest that Ionidamine and AF2785, which are open-channel blockers of CFTR at low concentrations, also affect CFTR gating at high concentrations. We conclude that these indazole compounds provide new pharmacological tools for the investigation of CFTR. By virtue of their interference with reproductive processes, these drugs have the potential for being developed into novel male contraceptives.