Tomato (Solanum lycopersicum) fruit ripening is characterized by a massive accumulation of carotenoids (mainly lycopene) as chloroplasts change to chromoplasts. To address the question of the role of sugars in controlling carotenoid accumulation, fruit pericarp discs (mature green fruits) were cultured in vitro in the presence of various sucrose concentrations. A significant difference in soluble sugar content was achieved depending on external sucrose availability. Sucrose limitation delayed and reduced lycopene and phytoene accumulation, with no significant effect on other carotenoids. Chlorophyll degradation and starch catabolism were not affected by variations of sucrose availability. The reduction of lycopene synthesis observed in sucrose-limited conditions was mediated through metabolic changes illustrated by reduced hexose accumulation levels. In addition, variations of sucrose availability modulated PSY1 gene expression. Taken together our results suggest that the modulation of carotenoid accumulation by sucrose availability occurs at the metabolic level and involves the differential regulation of genes involved in carotenoid biosynthesis.