Gastric vesicles purified from acid-secreting rabbit stomach display K⁺ permeability manifested by the valinomycin-independent proton pumping of H⁺-K⁺-ATPase as monitored by acridine orange quenching. This apparent K⁺ permeability is attenuated by the treatment of the membrane with 5 mM Mg²⁺, and this phenomenon has been attributed to membrane-bound phosphoprotein phosphatase. However, with the exception of the nonspecific inhibitor pyrophosphate, protein phosphatase inhibitors failed to inhibit the loss of K⁺ permeability. Preincubation of the membrane with neomycin, a phospholipase C inhibitor, surrogated the effect of Mg²⁺, whereas another inhibitor, U-73122, did not. Phosphatidylinositol 4,5-bisphosphate (PIP₂) restored the attenuated K⁺ permeability by treatment with either Mg²⁺ or neomycin. Furthermore, either phosphatidylinositol bound to phosphatidylinositol transfer protein or phosphatidylinositol 4,5,6-trisphosphate (PIP₃) surrogated the effect of PIP₂. Mg²⁺ and neomycin reduced K⁺ permeability in the membrane as determined by Rb⁺ influx and K⁺-dependent H⁺ diffusion. Treatment with Mg²⁺ reduced the contents of PIP₂ and PIP₃ in the membrane. These results suggest that PIP₂ and/or PIP₃ maintain K⁺ permeability, which is essential for proton pumping in the apical membrane of the secreting parietal cell.