As in many other fleshy fruits, the predominant organic acids in ripe peach (Prunus persica (L.) Batsch) fruit are malic and citric acids. The accumulation of these metabolites in fruit flesh is regulated during fruit development. Six peach fruit-related genes implicated in organic acid metabolism (mitochondrial citrate synthase; cytosolic NAD-dependent malate dehydrogenase, and cytosolic NADP-dependent isocitrate dehydrogenase) and storage (vacuolar proton translocating pumps: one vacuolar H+-ATPase, and two vacuolar H+-pyrophosphatases) were cloned. Five of these peach genes were homologous to genes isolated from fruit in other fleshy fruit species. Phylogenetic and expression analyses suggested the existence of a particular vacuolar pyrophosphatase highly expressed in fruit. The sixth gene was the first cytosolic NAD-dependent malate dehydrogenase gene isolated from fruit. Gene expression was studied during the fruit development of two peach cultivars, a normal-acid (Fantasia) and a low-acid (Jalousia) cultivar. The overall expression patterns of the organic acid-related genes appeared strikingly similar for the two cultivars. The genes involved in organic acid metabolism showed a stronger expression in ripening fruit than during the earlier phases of development, but their expression patterns were not necessarily correlated with the changes in organic acid contents. The tonoplast proton pumps showed a biphasic expression pattern more consistent with the patterns of organic acid accumulation, and the tonoplast pyrophosphatases were more highly expressed in the fruit of the low-acid cultivar during the second rapid growth phase of the fruit.