Intracellular Ca(2+) mobilization plays important roles in cell survival, proliferation, and differentiation of osteoblasts. In this study, we identified a novel type of Ca(2+)-activated K(+) channel in human osteoblasts and investigated its physiological roles. Using RT-PCR methods and single-channel analysis in the patch-clamp technique, we found that BK and IK channels were genetically expressed in human osteoblasts and had electrophysiological properties similar to those reported previously for the channels in other organs (conductance, voltage dependence, and sensitivity to intracellular Ca(2+)). Taking advantage of the fact that ATP induces elevation of the intracellular Ca(2+) concentration in human osteoblasts, we successfully demonstrated that ATP-induced hyperpolarization was effectively inhibited by the IK channel blockers charybdotoxin and clotrimazole and by a P2 purinergic receptor antagonist, suramin, but not by the BK channel blockers tetraethylammonium chloride and iberiotoxin under the current-clamp mode of whole-cell clamp. The present study is the first to demonstrate the electrophysiological properties and functional expression of IK channels in human osteoblasts, findings which suggest that IK channels are regulators of membrane potential that give rise to intracellular Ca(2+) mobilization by physiological stimulation.