The modulation of ion channel proteins by cellular redox potential has emerged recently as a significant determinant of channel function. We have investigated the influence of sulfhydryl redox reagents on human brain Ca²⁺-activated K⁺channels (hslo) expressed in both human embryonic kidney 293 cells and Xenopus oocytes using macropatch and single-channel analysis. Intracellular application of the reducing agent dithiothreitol (DTT): (1) shifts the voltage of half-maximal channel activation (V_0.5) ≈18 mV to more negative potentials without affecting the maximal conductance or the slope of the voltage dependence; (2) slows by ≈10-fold a time-dependent right-shift in V_0.5 values (“run-down”); (3) speeds macroscopic current activation kinetics by ≈33%; and (4) increases the single-channel open probability without affecting the unitary conductance. In contrast to DTT treatment, oxidation with hydrogen peroxide shifts macropatchV_0.5 values to more positive potentials, increases the rate of channel run-down, and decreases the single-channel open probability. K_Ca channels cloned fromDrosophila differ from hslo channels in that they show very little run-down and are not modulated by the addition of DTT. These data indicate that hsloCa²⁺-activated K⁺ channels may be modulated by changes in the cellular redox potential as well as by the transmembrane voltage and the cytoplasmic Ca²⁺concentration.