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 Mg(2+), 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 Mg(2+), whereas another inhibitor, U-73122, did not. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) restored the attenuated K(+) permeability by treatment with either Mg(2+) or neomycin. Furthermore, either phosphatidylinositol bound to phosphatidylinositol transfer protein or phosphatidylinositol 4,5,6-trisphosphate (PIP(3)) surrogated the effect of PIP(2). Mg(2+) and neomycin reduced K(+) permeability in the membrane as determined by Rb(+) influx and K(+)-dependent H(+) diffusion. Treatment with Mg(2+) reduced the contents of PIP(2) and PIP(3) in the membrane. These results suggest that PIP(2) and/or PIP(3) maintain K(+) permeability, which is essential for proton pumping in the apical membrane of the secreting parietal cell.