ATP-sensitive K+ (KATP) channels are hetero-octameric complexes of two subunits: four pore-forming Kir6.x and four regulatory SURx subunits. In vascular and non-vascular smooth muscle (SM), KATP channels contribute to the resting membrane potential and modulate the muscle tone. KATP channels in these tissues are comprised of the same auxiliary subunit (SUR2B) but different pore-forming subunits. Kir6.1 is predominantly found in vascular SM while Kir6.2 is found in non-vascular SM. In spite of the fact that Kir6.1 and Kir6.2 share ~70% sequence identity their sensitivity to ATP differs by more than 30-fold. These observations suggest that in vascular SM cells KATP channels are active under resting metabolic condition while in non-vascular SM the contribution of these channels to the membrane potential will become more relevant under conditions of high metabolic demand. Here we investigate the molecular basis for the differential response to ATP of Kir6.1 and Kir6.2-containing KATP channels. We performed systematic mutations in Kir6.2 exchanging residues with the reciprocal residues in Kir6.1. The putative ATP and PIP2 binding sites, “slide helix” and critical residues at the interface between subunits were investigated. Inside-out patch-clamp recordings from HEK293T cells heterologously expressing wild type and mutant Kir6.x co-expressed with SUR2B were used in this study. The extracellular solution contained (in mM): 140 KCl, 10 HEPES, 1.2 MgCl2 and 2.6 CaCl2; pH 7.4 with KOH. The intracellular solution contained (in mM): 115 KCl, 2 MgCl2, 1 CaCl2, 10 EGTA, 10 HEPES, 25 KOH; pH 7.2 with KOH. Mg-ATP was included as necessary and the pH was readjusted to 7.2 with KOH. All numerical data are presented as mean ± SEM. Mutations within the ATP binding site, slide helix and subunit interaction domains of Kir6.2 had no effect on ATP sensitivity. However, simultaneous mutation of two residues in Kir6.2 within the PIP2 binding site, K39S and N41A, caused an increase in IC50 for ATP from 101 ± 22µM (n=17), in wild-type channels, to 264 ± 55µM (n=16), bringing it towards that of Kir6.1. This is a small shift (~2.5-fold) compared to the difference observed between wild-type Kir6.1 and Kir6.2 (>30-fold). The effect of this mutation is not secondary to a change in channel gating as the single-channel open probability (Po) remained unchanged (0.44 ± 0.02, (n=6, Kir6.2-K39S-N41A) vs 0.38 ± 0.07, (n=6, Kir6.2)). Furthermore, maximal activation by PIP2 and the effect of repeated PIP2 application on ATP inhibition were unaffected. These data suggest the difference in ATP sensitivity between cloned vascular and non-vascular KATP channels is not due to distinct ATP-binding affinities/efficacies or the sensitivity to PIP2 and that other regions of the channels than those tested here must be involved in determining the difference in ATP sensitivity.