Ascorbic acid (AsA) is an essential phytonutrient, and tomatoes are an important dietary source. Tomato fruit AsA content is regulated by light conditions and is cultivar-dependent, but their regulations is unknown about the role of source-sink relationships. This study was designed to compare the light/dark-controlled regulation of AsA between intact (plant-attached fruits with source-sink relationships) and in vitro culture (plant-detached fruits without source-sink relationships) tomato fruits of five cultivars. Compared with fruits grown under light conditions, AsA content was lower in both intact and in vitro fruits grown in the dark; however, the decrease in AsA in the dark treatment was markedly lower in intact fruits (17-29%) than in vitro fruits (44-74%) and was dependent on the cultivar. Antioxidant enzyme activities and lipid peroxidation were lower under the dark condition in in vitro fruits (30-90%) but not intact fruits. Therefore, the reduction of AsA content under darkness occurred due to decreased in situ biosynthesis and antioxidant system activity in fruit without source-sink relationships. Furthermore, the reduction of AsA content induced by darkness was mitigated by source-sink relationships, as indicated by the intact fruit results, and their effects depend on cultivars.