Two electrode voltage clamp and single channel recordings were used to investigate the actions of various ATP-sensitive K(+) (KATP) channel inhibitors on cloned KATP channels, expressed in Xenopus oocytes and HEK 293 cells. Oocytes expressing Kir6.2 and SUR1 gave rise to inwardly rectifying K+ currents following bath application of 3 mM sodium azide. Inside-out recordings from non-azide treated oocytes demonstrated the presence of KATP channels which were activated by direct application of 3 mM azide and 0.1 mM Mg-ATP. Tolbutamide inhibited azide-induced macroscopic Kir6.2-SUR1 currents, recorded from Xenopus oocytes, with an IC50 value similar to native KATP channels. Ciclazindol and englitazone also inhibited these currents in a concentration-dependent manner, but with relative potencies substantially less than for native K(ATP) channels. Single channel currents recorded from inside-out patches excised from oocytes expressing Kir6.2-SUR1 currents were inhibited by tolbutamide, Mg-ATP, englitazone and ciclazindol, in the absence of azide, with potencies similar to native KATP channels. In the presence of azide, Kir6.2-SUR1 currents were inhibited by englitazone and tolbutamide but not ciclazindol. Single channel currents derived from Kir6.2?26, expressed in HEK 293 cells, were inhibited by ciclazindol and englitazone irrespective of the absence or presence of SUR1. In conclusion, heterologously expressed Kir6.2 and SUR1 recapitulate the pharmacological profile of native pancreatic beta-cell KATP channels. However, currents induced by azide exhibit a substantially reduced sensitivity to ciclazindol. It is likely that ciclazindol and englitazone inhibit KATP currents by interaction with the Kir6.2 subunit.