(1) We have investigated the possible roles of sulphonylurea receptor (SUR) type 1 and 2B in the activity of pig urethral ATP-sensitive K(+) channels (K(ATP) channels) by use of patch-clamp and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. (2) In voltage-clamp experiments, not only diazoxide, a SUR1 and weak SUR2B activator, but also pinacidil, a selective SUR2 activator, caused an inward current at a holding potential of -50 mV in symmetrical 140 mM K(+) conditions. (3) Gliclazide (</=1 micro M), a selective SUR1 blocker, inhibited the 10 micro M pinacidil-induced currents (K(i)=177 micro M) and the 500 micro M diazoxide-induced currents (high-affinity site, K(i1)=5 nM; low-affinity site, K(i2)=108 micro M) at -50 mV. (4) Application of tolbutamide (</=100 micro M) reversibly caused an inhibition of the 500 micro M diazoxide-induced current at -50 mV. (5) MCC-134, a SUR type-specific K(ATP) channel regulator (1-100 micro M), produced a concentration-dependent inward K(+) current, which was suppressed by the application of glibenclamide at -50 mV. The amplitude of the MCC-134 (100 micro M)-induced current was approximately 50% of that of the 100 micro M pinacidil-induced currents. (6) Using cell-attached configuration, MCC-134 activated a glibenclamide-sensitive K(ATP) channel which was also activated by pinacidil. (7) RT-PCR analysis demonstrated the presence of SUR1 and SUR2B transcripts in pig urethra. 8 These results indicate that both SUR1 and SUR2B subunits play a functional role in regulating the activity of pig urethral K(ATP) channels and that SUR1 contributes less than 25% to total K(ATP) currents.