1. The sulphonylurea drug glibenclamide is a widely used inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR). To investigate how glibenclamide inhibits CFTR, we studied CFTR Cl- channels using excised inside-out membrane patches from cells expressing wild-type human CFTR. 2. Addition of glibenclamide (10-100 microM) to the intracellular solution caused a concentration-dependent decrease in the open time of CFTR Cl- channels, but closed times did not change. This suggests that glibenclamide is an open-channel blocker of CFTR. 3. Glibenclamide is a weak organic acid. Acidification of the intracellular solution relieved glibenclamide inhibition of CFTR, suggesting that the anionic form of glibenclamide inhibits CFTR. 4. To begin to identify the glibenclamide binding site in CFTR, we investigated whether glibenclamide competes with either MgATP or Cl- ions for a common binding site. Glibenclamide inhibition of CFTR was unaffected by nucleotide-dependent stimulation of CFTR, suggesting that glibenclamide and intracellular MgATP interact with CFTR at distinct sites. 5. Glibenclamide inhibition of CFTR was voltage dependent and enhanced when the external Cl- concentration was decreased. The data suggest that glibenclamide and Cl- ions may compete for a common binding site located within a large intracellular vestibule that is part of the CFTR pore.