The carrier-mediated, electroneutral exchange of Na⁺ for H⁺ across the plasma membrane does not directly consume metabolic energy. Nevertheless, acute depletion of cellular ATP markedly decreases transport. We analyzed the possible involvement of polyphosphoinositides in the metabolic regulation of NHE1, the ubiquitous isoform of the Na⁺/H⁺ exchanger. Depletion of ATP was accompanied by a marked reduction of plasmalemmal phosphatidylinositol 4,5-bisphosphate (PIP₂) content. Moreover, sequestration or hydrolysis of plasmalemmal PIP₂, in the absence of ATP depletion, was associated with profound inhibition of NHE1 activity. Examination of the primary structure of the COOH-terminal domain of NHE1 revealed two potential PIP₂-binding motifs. Fusion proteins encoding these motifs bound PIP₂ in vitro. When transfected into antiport-deficient cells, mutant forms of NHE1 lacking the putative PIP₂-binding domains had greatly reduced transport capability, implying that association with PIP₂ is required for optimal activity. These findings suggest that NHE1 activity is modulated by phosphoinositides and that the inhibitory effect of ATP depletion may be attributable, at least in part, to the accompanying net dephosphorylation of PIP₂.