Modulation of the cloned human intermediate-conductance Ca 2 + -activated K + channel (hIK) by the compound 1-ethyl-2-benzimidazolinone (EBIO) was studied by patch-clamp technique using human embryonic kidney cells (HEK 293) stably expressing the hIK channels. In whole-cell studies, intracellular concentrations of free Ca 2 + were systematically varied, by buffering the pipette solutions. In voltage-clamp, the hIK specific currents increased gradually from 0 to ~300 pA/pF without reaching saturation even at the highest Ca 2 + concentration tested (300 nM). In the presence of EBIO (100 μM), the Ca 2 + -activation curve was shifted leftwards, and maximal currents were attained at 100 nM Ca 2 + . In current-clamp, steeply Ca 2 + -dependent membrane potentials were recorded and the cells gradually hyperpolarised from -20 to -85 mV when Ca 2 + was augmented from 0 to 300 nM. EBIO strongly hyperpolarised cells buffered at intermediate Ca 2 + concentrations. In contrast, no effects were detected either below 10 nM (no basic channel activation) or at 300 nM Ca 2 + (V m close to E K ). Without Ca 2 + , EBIO-induced hyperpolarisations were not obtainable, indicating an obligatory Ca 2 + -dependent mechanism of action. When applied to inside-out patches, EBIO exerted a Ca 2 + -dependent increase in the single-channel open-state probability, showing that the compound modulates hIK channels by a direct action on the α-subunit or on a closely associated protein. In conclusion, EBIO activates hIK channels in whole-cell and inside-out patches by a direct mechanism, which requires the presence of internal Ca 2 + .