Changes in metabolites were studied during the fruit development of two greenhouse grown peach [Prunus persica (L.) Batsch] cultivars with low acidity (`Jalousia') or normal acidity (`Fantasia'). Both cultivars had the same sucrose concentration in fruit mesocarp at maturity. In the fruit juice, pH was higher and titratable acidity was lower for `Jalousia' than for `Fantasia' from 80 days after bloom to maturity. At four different times during fruit development, in vivo 13C NMR spectroscopy was used to measure the vacuolar pH of fruit mesocarp. At 55 days after bloom, the vacuolar pH of fruit mesocarp was not significantly different between `Jalousia' and `Fantasia', whereas the juice pH was different between cultivars. The three major organic acids in fruit mesocarp were malic, citric, and quinic acids for both cultivars. Citric acid concentrations were similar in both cultivars until ≈85 days after bloom and then became significantly higher in `Fantasia'. A significantly higher concentration in malic acid in `Fantasia' than in `Jalousia' was observed from the end of the first growth phase to maturity. At maturity, `Fantasia' fruit had two and five times more malic and citric acid, respectively, than `Jalousia' fruit. The differences observed between `Jalousia' and `Fantasia' fruit for malic and citric acid concentrations accounted for the difference in titratable acidity. The differences in acid concentration appeared during the plateau between the two rapid growth phases of the fruit, i.e., far before the onset of maturation. The three major amino acids were asparagine, glutamic acid, and proline for both cultivars. Their concentration followed similar patterns in acid and low-acid fruit.