ATP-sensitive K⁺ (K_ATP) channels, composed of pore-forming Kir6.2 and regulatory SURx subunits, are critical for the physiological function of many tissues by acting as metabolic sensors. Metabolic regulation of K_ATP channel activity is achieved by changes in the intracellular concentrations of adenine nucleotides. Binding of ATP to Kir6.2 inhibits K_ATP channels, while interaction of Mg-nucleotides with SUR1 promotes channel opening. Recently, a mutation in Kir6.2 (F333I) that enhances the activatory effect of SUR1, has been reported (Tammaro et al. 2005). We used this mutation as a tool to analyse the activation properties of different SUR isoforms that contribute to the varied metabolic sensitivities of K_ATP channels containing SUR1, SUR2A and SUR2B subunits. Macroscopic K_ATP currents were recorded from inside-out patches from Xenopus laevis oocytes 1-3 days after injection with Kir6.2-F333I and SURx mRNAs. The pipette solution contained (mM): 140 KCl, 1.2 MgCl₂, 2.6 CaCl₂, 10 Hepes (pH 7.4) plus various ATP or ADP concentrations. The Mg-free internal (bath) solution contained (mM): 107 KCl, 1 K₂SO₄, 10 EGTA, 10 Hepes (pH 7.2). The Mg-containing solution consisted of Mg-free solution plus 2mM MgCl₂ and MgATP or MgADP (instead of ATP or ADP). Values are means±S.E.M. In the absence of Mg²⁺, only small differences were seen in the IC₅₀ for ATP inhibition of SUR1 (211±28μM, n=6), SUR2A (128±9μM, n=4) and SUR2B (119±19μM, n=4) containing channels. However, in the presence of Mg²⁺, SUR2A currents were blocked by ATP (IC₅₀ = 541±81μM, n=5) whereas SUR1 or SUR2B currents were activated (by ~5 and ~1.5 fold in 1 mM MgATP, respectively). The stimulatory effect of MgATP on SUR1 channels was abolished by mutations that prevent MgATP binding/hydrolysis. Both SUR2A and SUR2B currents were activated by 1 mM MgADP: 5.5±1.0 (n=5) and 5.0±0.5 (n=5) fold, respectively. The fact that SUR2A exhibits activation by MgADP but not by MgATP suggests that ATP binding/hydrolysis may be less than that of SUR1 or SUR2B. The differences in nucleotide handling by the various SUR isoforms may help explain why K_ATP channels are active in pancreatic beta cells over a wide range of metabolic conditions while in smooth muscle or the heart they open only under hypoxia or metabolic inhibition.