Multiple types of ATP-sensitive potassium (K(ATP)) channels have been described in smooth muscle, including those inhibited by ATP and those activated by nucleotide diphosphate (K(NDP)). The molecular identities of these channels have been proposed to be SUR2B/Kir6.2 and SUR2B/Kir6.1, respectively. However, subunit expression is largely unknown in vascular muscle, and the native channel has not been reported previously in human tissue. We used the patch-clamp technique to examine K(ATP) channel properties in cultured human pulmonary artery smooth muscle cells (HPASMC). Under physiological recording conditions, levcromakalim (10 microM) hyperpolarized cells (approximately 25-30 mV) and activated a glibenclamide-sensitive, background K(+) current, which was smaller in proliferating cells. Lowering ATP from 1 to 0.1 mM significantly enhanced responses to levcromakalim in HPASMC but not in HEK-293 cells stably transfected with SUR2B/Kir6.1. In both cell types, levcromakalim activated a 28-29 pS channel, which, upon patch excision, required the presence of nucleotide diphosphate for significant openings. Transcripts for SUR2B and Kir6.1, but not Kir6.2, were found by reverse transcription-polymerase chain reaction in HPASMC and in rat pulmonary arterial tissue. We conclude that K(ATP) channels are expressed in human pulmonary artery, and whereas data are consistent with the presence of nucleotide diphosphate-activated potassium channels, native whole-cell regulation cannot be reconstituted fully in heterologous expression systems.