1. We have investigated the mechanism of action of the novel anti-diabetic agent mitiglinide (S 21403) on Kir6.2/SUR1, Kir6.2/SUR2A and Kir6.2/SUR2B types of ATP-sensitive potassium (K(ATP)) channel. These possess a common pore-forming subunit, Kir6.2, and different regulatory sulphonylurea receptor (SUR) subunits. It is believed that they correspond to native K(ATP) channels in pancreatic beta-cells, heart and non-vascular smooth muscle, respectively. 2. Kir6.2 was coexpressed with SUR1, SUR2A or SUR2B in Xenopus oocytes and macroscopic currents were recorded in giant inside-out membrane patches. Mitiglinide was added to the intracellular membrane surface. 3. Mitiglinide inhibited Kir6.2/SUR currents at two sites: a low-affinity site on Kir6.2 and a high-affinity site on SUR. Low-affinity inhibition was similar for all three types of K(ATP) channel but high-affinity inhibition was greater for Kir6.2/SUR1 currents (IC(50), 4 nM) than for Kir6.2/SUR2A or Kir6.2/SUR2B currents (IC(50), 3 and 5 microM, respectively). 4. Inhibition of Kir6.2/SUR1 currents was only slowly reversible on the time scale of electrophysiological experiments. 5. Kir6.2/SUR1-S1237Y currents, which previously have been shown to lack high affinity tolbutamide inhibition, resembled Kir6.2/SUR2 currents in being unaffected by 100 nM but blocked by 10 microM mitiglinide. 6. Our results show that mitiglinide is a high-affinity drug that shows a 1000 fold greater affinity for the beta-cell type than the cardiac and smooth muscle types of K(ATP) channel, when measured in excised patches.