Aims/hypothesis. To investigate the tissue specificity of gliclazide for cloned beta-cell, cardiac and smooth muscle ATP-sensitive K-channels (K_ATP channels). These channels share a common pore-forming subunit, Kir6.2, which associates with different sulphonylurea receptor isoforms (SUR1 in beta-cells, SUR2A in heart, SUR2B in smooth muscle). Methods. Kir6.2 was coexpressed with SUR1, SUR2A or SUR2B in Xenopus oocytes, and channel activity was measured by recording macroscopic currents in giant inside-out membrane patches. Gliclazide was added to the intracellular membrane surface. Results. We reported previously that Kir6.2-SUR1 currents are blocked at two sites by tolbutamide: a high-affinity site on SUR1 and a low-affinity site on Kir6.2. We now show that gliclazide also inhibits beta-cell K_ATP channels at two sites: a high-affinity site, which is half-maximally blocked (K _i) at 50 ± 7 nmol/l (n = 8) and a low-affinity site with a K _i of 3.0 ± 0.6 mmol/l (n = 4). The high-affinity site on SUR1 was thus about 40-fold more sensitive to gliclazide than to tolbutamide (K _i∼ 2 μmol/l). Cloned cardiac and smooth muscle K_ATP channels did not show high-affinity block by gliclazide. Kir6.2-SUR2A currents exhibited a single low-affinity site with a K _i of 0.8 ± 0.1 mmol/l (n = 5), which is likely to reside on the Kir6.2 subunit. Conclusion/interpretation. Our results show that gliclazide is a sulphonylurea with high affinity and strong selectivity for the beta-cell type of K_ATP channel. [Diabetologia (1999) 42: 845–848]