KATP channels are thought to play an important role in the regulation of vascular tone. More specifically, Kir6.1/SUR2B has been implicated as the vascular KATP channel. The study of mice with a global genetic deletion of Kir6.1 and SUR2 has suggested a more complex picture for the role of KATP channels in vascular regulation than has been proposed (1,2). To address this, we have generated a murine model in which it is possible to conditionally delete the Kir6.1 subunit in smooth muscle (SM). Exon 2 of the mouse KCNJ8 gene was targeted. The targeting construct was transfected into ES cells derived from 129P2/Ola mice. One positive clone was isolated, injected into C57Bl/6J blastocysts and then implanted into pseudo-pregnant females (under Xylazine (6.6mg/ml) + ketamine (16.6mg/ml) I.P. anaesthesia). A number of male chimaeras were bred with Flp deleter mice to generate Kir6.1 (+, flx) mice. CreloxP technology allowed the tissue specific deletion of the gene by crossing the Kir6.1 (+, flx) mice with a cre line that expresses the recombinase in vascular SM (smMHC cre or sm22α cre). Tissue specific deletion was verified using PCR, Real-time RT-PCR, whole tissue organ bath studies and whole-cell patch clamp using acutely isolated aortic SM cells. Data are expressed as mean ± S.E.M., Student’s t test was used for statistical analysis. PCR on genomic DNA isolated from tail snips, SM and heart from smMHCcre+ Kir6.1 (flx, flx), sm22cre+ Kir6.1 (flx, flx) and control animals confirmed genetic deletion of exon 2 in SM but not in the tail or heart in both lines. Real-time RT-PCR showed substantially reduced expression of the transcript in SM in smMHCcre+ Kir6.1 (flx, flx) (93.17 ± 0.01 %, n=5, P<0.05) and sm22cre+ Kir6.1 (flx, flx) (95.13 ± 0.03 %, n=3, P<0.05) mice compared to control animals. Expression levels in the heart and brain of both lines were not significantly different from those in control tissues (P>0.1). Whole-cell patch clamp recordings from control single aortic SM cells showed that there was a K+ selective current activated by the KATP channel opener levcromakalim (39.85 ± 6.27 pA/pF compared to control; 20.84 ± 2.84 pA/pF, n=10, P<0.05) and inhibited by glibenclamide (15.25 ± 3.2 pA/pF, n=10, P<0.01). In contrast, cells from smMHCcre+ Kir6.1 (flx, flx) (control: 18.75 ± 1.86, levcromakalim: 19.35 ± 3.03, glibenclamide: 14.14 ± 1.54 pA/pF, n=10, P>0.05) and sm22cre+ Kir6.1 (flx, flx) (control: 18.75 ± 1.86, levcromakalim: 19.35 ± 3.03, glibenclamide: 14.14 ± 1.54 pA/pF, n=8, P>0.05) mice presented no such KATP current. Furthermore, organ bath studies in these mice showed that constricted aortic rings no longer relaxed in response to levcromakalim and had no response to glibenclamide. The tissue specific Kir6.1 knockout mouse will enable a better understanding of how KATP channel regulation integrates into whole animal cardiovascular physiology and pathology.