Hair disorders cause psychological distress but are generally poorly controlled; more effective treatments are required. Despite the long-standing use of minoxidil for balding, its mechanism is unclear; suggestions include action on vasculature or follicle cells. Similar drugs also stimulate hair, implicating ATP-sensitive potassium (K_ATP) channels. To investigate whether K_ATP channels are present in human follicles, we used organ culture, molecular biological, and immunohistological approaches. Minoxidil and tolbutamide, a K_ATP channel blocker, opposed each other’s effects on the growing phase (anagen) of scalp follicles cultured in media with and without insulin. Reverse transcriptase-polymerase chain reaction identified K_ATP channel component gene expression including regulatory sulfonylurea receptors (SUR) SUR1 and SUR2B but not SUR2A and pore-forming subunits (Kir) Kir6.1 and Kir6.2. When hair bulb tissues were examined separately, epithelial matrix expressed SUR1 and Kir6.2, whereas both dermal papilla and sheath exhibited SUR2B and Kir6.1. Immunohistochemistry demonstrated similar protein distributions. Thus, human follicles respond biologically to K_ATP channel regulators in culture and express genes and proteins for two K_ATP channels, Kir6.2/SUR1 and Kir6.1/SUR2B; minoxidil only stimulates SUR2 channels. These findings indicate that human follicular dermal papillae contain K_ATP channels that can respond to minoxidil and that tolbutamide may suppress hair growth clinically; novel drugs designed specifically for these channels could treat hair disorders.—Shorter, K., Farjo, N. P., Picksley, S. M., Randall, V. A. Human hair follicles contain two forms of ATP-sensitive potassium channels, only one of which is sensitive to minoxidil.