We identified and characterized Vnx1p, a novel vacuolar monovalent cation/H+ antiporter encoded by the open reading frame YNL321w from Saccharomyces cerevisiae. Despite the homology of Vnx1p with other members of the CAX (calcium exchanger) family of transporters, Vnx1p is unable to mediate Ca2+ transport but is a low affinity Na+/H+ and K+/H+ anti-porter with a Km of 22.4 and 82.2 mm for Na+ and K+, respectively. Sequence analyses of Vnx1p revealed the absence of key amino acids shown to be essential for Ca2+/H+ exchange. vnx1Delta cells displayed growth inhibition when grown in the presence of hygromycin B or NaCl. Vnx1p activity was found in the vacuoles and shown to be dependent on the electrochemical potential gradient of H+ generated by the action of the V-type H+-ATPase. The presence of Vnx1p at the vacuolar membrane was further confirmed with cells expressing a VNX1::GFP chimeric gene. Similar to Nhx1p, the prevacuolar compartment-bound Na+/H+ antiporter, the vacuole-bound Vnx1p appears to play roles in the regulation of ion homeostasis and cellular pH.