Several transporting epithelia in vertebrates and invertebrates contain cells that are specialized for proton or bicarbonate secretion. These characteristic ‘mitochondria-rich’ (MR) cells have several typical features, the most important of which is an extremely high expression of a vacuolar-type proton-pumping ATPase (H⁺V-ATPase) both on intracellular vesicles and on specific domains of their plasma membrane. Physiological modulation of proton secretion is achieved by recycling the H⁺V-ATPase between the plasma membrane and the cytoplasm in a novel type of nonclathrin-coated vesicle. In the kidney, these cells are involved in urinary acidification, while in the epididymis and vas deferens they acidify the luminal environment to allow normal sperm development. Osteoclasts are non-epithelial MR cells that use H⁺V-ATPase activity for bone remodeling. In some insects, similar cells in the midgut energize K⁺ secretion by means of a plasma membrane H⁺V-ATPase. This review emphasizes important structural and functional features of proton-secreting cells, describes the tissue distribution of these cells and discusses the known functions of these cells in their respective epithelia.