This study was designed to identify the single-channel properties and molecular entity of ATP-sensitive K(+) (K(ATP)) channels in guinea pig gastric myocytes with patch-clamp recording and RT-PCR. Pinacidil and diazoxide activated K(ATP) currents in a glibenclamide-sensitive manner. The open probability of channels was enhanced by the application of 10 microM pinacidil from 0.085 +/- 0.04 to 0.20 +/- 0.05 (n = 7) and was completely blocked by 10 microM glibenclamide. Single-channel conductance was 37.3 +/- 2.5 pS (n = 4) between -80 and -20 mV in symmetrical K(+) gradient conditions. In inside-out mode, K(ATP) channels showed no spontaneous openings and were activated by the application of nucleotide diphosphates to the cytoplasmic side. These single-channel properties are similar to those of the nucleotide diphosphate-dependent K(+) channels in vascular smooth muscle, which are composed of Kir6.1 and sulfonylurea receptor (SUR)2B. RT-PCR demonstrated the presence of Kir6.1, Kir6.2, and SUR2B in guinea pig stomach smooth muscle cells. These results suggest that K(ATP) channels in smooth muscle cells of the guinea pig stomach are composed of Kir6.1 and SUR2B.