K_ATP channels of pancreatic B-cells are completely inhibited in cell-free patches by physiological cytosolic ATP concentrations ([ATP]_c). However, insulin secretion in intact cells is regulated by changes in K_ATP channel activity. Since a metabolic barrier insulates K_ATP channels from [ATP]_c in cardiac myocytes, we postulated that K_ATP channels in B-cells may be positively regulated by an adenylate kinase (AK). B-cells were isolated from C57Bl6 mice. Single K_ATP channel currents were measured in the open cell attached (oca) or inside/out (i/o) mode of the patch-clamp technique. Western blots and immuno staining experiments were used to identify the AK. Comparing the open probability (P_o) of K_ATP currents measured in the i/o or oca mode, revealed a clear left-shift in the ATP dependence in cell-free patches. This points at an influence of cytosolic enzymes on the ATP block. Accordingly, AMP (0.1 mM) increased the P_o of all channels in the oca patch (NP_o) in the presence of 0.125 mM ATP (656±133 %; n=5). AMP was without an effect in oca patches where AK was inhibited by Ap5A (20 mM, n=4) or in i/o patches (n=5). The AK isoform 1 (AK1) was detected by Western blot analysis of whole islets. This was confirmed by immunofluorescence images showing a co-localization of AK1 with insulin. We conclude that an AK, probably AK1, is part of a metabolic barrier protecting K_ATP channels of pancreatic B-cells from high [ATP]_c.