ATP-dependent K+ (K(ATP)) channel was purified from the inner mitochondrial membrane and reconstituted into the bilayer lipid membrane. The K(ATP) activity was inhibited by high concentrations of ATP and ADP, but was activated by low concentrations (up to 200 microM) of ADP. p-Diethylaminoethyl benzoate (DEB) acted as a K(ATP) opener: at micromolar concentrations, it reversed the inhibitory effect of ATP and ADP, and also prevented K(ATP) rundown. Pelargonidine extracted from Pelargonium flowers reduced the spontaneous activity of K(ATP) channels and attenuated DEB-induced channel activation. The opposite action of DEB and pelargonidine on K(ATP) corresponded to the opposite redox properties of the two agents in reactions with free radicals: DEB behaved as an electron donor, while pelargonidine acted as an electron acceptor. We suggest that the thiol groups in mitochondrial K(ATP) are targets for redox-active ligands.