Mitochondria and inward rectifier K⁺ channels are both impaired in failing myocardium. We previously showed that K_ir2 inward rectifier K⁺ channels are inhibited by agents that affect mitochondria, and that K_ir2.2 and K_ir2.3 are more sensitive than K_ir2.1^{1, 2}. The sensitivity of K_ir2.1 channels to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) is increased by mutation of His53 to Gln (the equivalent residue in K_ir2.2 and K_ir2.3), Ala or Glu, but not Arg or Lys³. Neutralisation of His53 decreases the affinity of K_ir2.1 for phosphatidylinositol bisphosphate (PIP₂), a positive regulator of K_ir2 channels⁴, raising the possibility that FCCP inhibits K_ir2 channels via PIP₂ depletion. Such a mechanism may be reversible in intact cells but not in excised membrane patches in conditions that preclude PIP₂ regeneration. Treatment of Xenopus oocytes expressing K_ir2.2 with 10 µM FCCP for 105 min inhibited inward rectifier current to 7.2±1.8% (mean±sem) of control (p<.0001; n=5). Removal of FCCP resulted in recovery within 26.5 hr. K_ir2.2 current in cell-attached patches was 148±49 pA (n=13) in FCCP-treated oocytes and 1689±495 pA (n=15) in untreated oocytes (p<.01). K_ir2.2 current increased upon excision and perfusion of inside-out patches from FCCP-treated oocytes. However, this current run-up did not reflect recovery from inhibition by FCCP because run-up also occurred in inside-out patches from untreated oocytes. The fraction of total current that increased after patch excision was 0.79±0.05 in control and 0.89±0.03 in FCCP-treated oocytes (p=0.1; n=6). These results are consistent with a PIP₂ depletion mechanism of FCCP action. Run-up in inside-out patches suggests a soluble inhibitory factor in Xenopus oocytes. This was supported by experiments in which K_ir2.2 current was re-inhibited by ‘cramming’ inside-out patches into the oocyte; p<.05 for cell-attached vs maximum inside-out (I_max), p>.05 for cell-attached vs crammed patch, p<.05 for I_max vs crammed patch (n=8; repeated measures ANOVA). In summary, (1) FCCP inhibits K_ir2.2 by a mechanism that may involve PIP₂ depletion; (2) recovery requires intracellular components that are not membrane-bound; (3) Xenopus oocytes possess an endogenous soluble factor that inhibits K_ir2.2. Statistics: unpaired t-test unless otherwise stated.