Specific recognition of phosphatidylinositol 3-phosphate [PtdIns(3)P] by the FYVE domain targets cytosolic proteins to endosomal membranes during key signaling and trafficking events within eukaryotic cells. Here, we show that this membrane targeting is regulated by the acidic cellular environment. Lowering the cytosolic pH enhances PtdIns(3)P affinity of the FYVE domain, reinforcing the anchoring of early endosome antigen 1 (EEA1) to endosomal membranes. Reversibly, increasing the pH disrupts phosphoinositide binding and leads to cytoplasmic redistribution of EEA1. pH dependency is due to a pair of conserved His residues, the successive protonation of which is required for PtdIns(3)P head group recognition as revealed by NMR. Substitution of the His residues abolishes PtdIns(3)P binding by the FYVE domain in vitro and in vivo. Another PtdIns(3)P-binding module, the PX domain of Vam7 and p40 phox is shown to be pH-independent. This provides the fundamental functional distinction between the two phosphoinositide-recognizing domains. The presented mode of FYVE regulation establishes the unique function of FYVE proteins as low pH sensors of PtdIns(3)P and reveals the critical role of the histidine switch in targeting of these proteins to endosomal membranes.phosphoinositide ͉ early endosome antigen 1 T he specific recruitment of FYVE proteins to endosomes, multivesicular bodies, and phagosomes is primarily mediated by FYVE domain binding to membrane-embedded phosphatidylinositol 3-phosphate [PtdIns(3)P] (1-3). Additional membrane anchoring is provided by hydrophobic insertion into the bilayer, electrostatic interactions with acidic lipids (4, 5), and dimerization (6, 7). These synergistic factors are thought to be largely responsible for directing FYVE proteins to PtdIns(3)P-enriched membranes. Yet, the current model of FYVE domain function has several limitations and is not predictive. Some FYVE proteins localize inexplicably to sites that contain little PtdIns(3)P, such as the Golgi and endoplasmic reticulum (8, 9). On the other hand, places with significant PtdIns(3)P concentrations, such as the nucleus and mitochondria (10), do not appear to attract FYVE proteins. All canonical FYVE domains have been found to target PtdIns(3)P (refs. 6, 10-16); however, their binding properties were investigated at pH values of 6.5-8.0, leading to a discrepancy in estimated affinities despite the highly conserved sequences and structures of the FYVE proteins.The FYVE domain is defined by the three conserved elements: the N-terminal WxxD, the central RR͞KHHCR, and the Cterminal RVC motifs that form the PtdIns(3)P binding site seen in the solution and crystal structures of early endosome antigen 1 (EEA1), Hrs, and Vps27p FYVE (6,13,17,18). The PtdIns(3)P headgroup is coordinated by a cluster of four Arg͞Lys and two His residues and the N-terminal Asp. Among them, the two adjacent His residues are the most conserved in the FYVE domain sequences. In this work, we demonstrate that anchoring of the FYVE domain to PtdIns(3)P-enriched ...