The protonation state of soluble and membrane-associated macromolecules dictates their charge, conformation and functional activity. In addition, protons (H+, or their equivalents) partake in numerous metabolic reactions and serve as a source of electrochemical energy to drive the transmembrane transport of both organic and inorganic substrates. Stringent regulation of the intracellular pH is therefore paramount to homeostasis. While the regulation of the cytosolic pH has been studied extensively, our understanding of the determinants of the [H+] of intracellular organelles has developed more slowly, limited by their small size and inaccessibility. Recently, however, targeting of molecular probes to the organellar lumen, together with advances in genomic, proteomic and electrophysiological techniques have led to the identification and characterization of unique pumps, channels and transporters responsible for the establishment and maintenance of intraorganellar pH. These developments and their implications to cellular function in health and disease are the subject of this review.