Potassium channels have been involved in epithelial tumorigenesis but the role of small-conductance Ca(2+)-activated K(+) channels is unknown. We report here that small-conductance Ca(2+)-activated K(+) channels are expressed in a highly metastasizing mammary cancer cell line, MDA-MB-435s. Patch-clamp recordings showed typical small-conductance Ca(2+)-activated K(+) channel-mediated currents sensitive to apamin, 4-aminopyridine, and tetraethylammonium. Moreover, the cells displayed a high intracellular calcium concentration, which was decreased after 24 hours of apamin treatment. By regulating membrane potential and intracellular calcium concentration, these channels were involved in MDA-MB-435s cell migration, but not in proliferation. Only SK3 protein expression was observed in these cells in contrast to SK2, which was expressed both in cancer and noncancer cell lines. Whereas small interfering RNA directed against SK3 almost totally abolished MDA-MB-435s cell migration, transient expression of SK3 increased migration of the SK3-deficient cell lines, MCF-7 and 184A1. SK3 channel was solely expressed in tumor breast biopsies and not in nontumor breast tissues. Thus, SK3 protein channel seems to be a new mediator of breast cancer cell migration and represents a potential target for a new class of anticancer agents. [Mol Cancer Ther 2006;5(11):2946-53].