Blackcurrant (Ribes nigrum L.) is a widely grown commercial crop valued for its high vitamin C (l-ascorbic acid, AsA) content. In the present study, a systematic analysis of the mechanism of fruit AsA accumulation was undertaken. AsA accumulation occurred during fruit expansion and was associated with high in situ biosynthetic capacity via the l-galactose pathway and low rates of turnover. Cessation of AsA accumulation was associated with reduced biosynthesis and increased turnover. Translocation of AsA from photosynthetic or vegetative tissues contributed little to fruit AsA accumulation. Manipulation of substrate availability by defoliation had no effect on fruit AsA concentration but significantly reduced fruit yields. Supply of the AsA precursor l-galactono-1,4-lactone to intact, attached fruit transiently increased fruit AsA concentration which rapidly returned to control levels after removal of the compound. These data suggest strong developmental, metabolic and genetic control of AsA accumulation in blackcurrant fruit and indicate the potential for breeding high AsA cultivars.