V-ATPase-driven intra-vesicular acidification is crucial for vesicular trafficking. Defects in vesicular acidification and trafficking have recently been recognized as essential determinants of various human diseases. An important role of endosomal acidification in receptor-ligand dissociation and in activation of lysosomal hydrolytic enzymes is well established. However, the molecular mechanisms by which lumenal pH information is transmitted to the cytosolic small GTPases that control trafficking events such as budding and coat formation are unknown. Here we discuss our recent discovery that endosomal V-ATPase is a pH-sensor regulating the degradative pathway (1). We also propose the hypothetical molecular mechanism involved. Previously, work from Schulz and colleagues reported a pH-dependent interaction of Arf small GTPases with purified microsomal vesicles (2). However, the specific members of the Arf-family that are involved in this recruitment were not established. Also, the cellular compartment in which this interaction took place and the mechanism of pH-dependent Arf recruitment remained unknown. Subsequent studies from Gruenberg and colleagues demonstrated acidification-dependent recruitment of COP and Arf1 proteins onto early endosomes (3). These authors proposed that a hypothetical endosomal trans-membrane pH sensor is involved in a direct interaction with these proteins and that this biochemical event is necessary for the formation of transport carrier vesicles. In addition, the presence of a pH sensor in yeast vacuoles has been recently proposed (4). While the existence of a vesicular pH sensor was suggested in all these studies, its nature and pH-sensing molecular mechanism remained obscure. Kidney proximal tubule (PT) cells have an extensive endocytic apparatus that is critical for the reabsorption and degradation of filtered proteins via the endosomal/lysosomal pathway. The acidification of PT endosomes and lysosomes is driven by V-ATPase. The importance of endosomal acidification is underlined by our finding that V-ATPase inhibitors and acidification uncouplers strongly abolish function of this pathway (1). Recent data from our laboratory provide new insights into the regulation of this process by trans-membrane V-ATPase and cytosolic small GTPases. In particular, we have demonstrated that Arf6 and ARNO are targeted to early endosomes and colocalize with V-ATPase (5). Moreover, specific recruitment of ARNO and Arf6 (but not Arf1) from the cytosol to endosomes depends upon V-ATPase-driven intra-endosomal acidification, so implicating this biochemical event in regulating the protein degradative pathway. Importantly, our study also postulated the existence of a trans-membrane pH-sensing protein (PSP) in early endosomes and its direct interaction with ARNO and Arf6 (5). However, the nature of PSP and the precise mechanism of its pH-dependent interaction with small GTPases remained illusive. In search of the pH-sensing protein, we recently examined the role of V-ATPase and small GTPases in the trafficking of albumin-Alexa594 via the PT protein degradative pathway (1). We found that inhibition of endosomal acidification by bafilomycin selectively affects the degradative pathway by preventing the delivery of albumin-Alexa594 from early to late endosomes. We also showed that the trans-membrane a2-isoform of V-ATPase is specifically targeted to early endosomes. Its cytosolic N-terminal tail directly interacts with ARNO and the trans-membrane c-subunit of V-ATPase interacts with Arf6. Importantly, the interaction between the V-ATPase a2-isoform and ARNO is modulated by acidification of the endosomal lumen and regulates the protein degradative pathway. These results led us to propose that V-ATPase itself might be the long sought-after PSP. According to our model V-ATPase is responsible for: i) the generation of a pH gradient between vesicular membranes; ii) sensing of intra-vesicular pH; and iii) transmitting this information to the cytosolic side of the membrane. Hypothesis: Based on extensive experimental evidence on the crucial role of histidine residues in the function of pH-sensing proteins in eukaryotic cells, we hypothesize that pH-sensitive histidines within the intra-endosomal loops and C-terminal luminal tail of the a2-subunit of V-ATPase could also be involved in the pH-sensing function of V-ATPase. The crucial role of these histidine residues in pH-dependent conformational changes of the V-ATPase a2-isoform, its interaction with ARNO and ultimately in its acidification-dependent regulation of the endosomal/lysosomal protein degradative pathway remain to be demonstrated.