The color of tomato fruit is mainly determined by carotenoids and flavonoids. Phenotypic analysis of an introgression line (IL) population derived from a cross between Solanum lycopersicum 'Moneyberg' and the wild species Solanum chmielewskii revealed three ILs with a pink fruit color. These lines had a homozygous S. chmielewskii introgression on the short arm of chromosome 1, consistent with the position of the y (yellow) mutation known to result in colorless epidermis, and hence pink-colored fruit, when combined with a red flesh. Metabolic analysis showed that pink fruit lack the ripening-dependent accumulation of the yellow-colored flavonoid naringenin chalcone in the fruit peel, while carotenoid levels are not affected. The expression of all genes encoding biosynthetic enzymes involved in the production of the flavonol rutin from naringenin chalcone was downregulated in pink fruit, suggesting that the candidate gene underlying the pink phenotype encodes a regulatory protein such as a transcription factor rather than a biosynthetic enzyme. Of 26 MYB and basic helix-loop-helix transcription factors putatively involved in regulating transcription of genes in the phenylpropanoid and/or flavonoid pathway, only the expression level of the MYB12 gene correlated well with the decrease in the expression of structural flavonoid genes in peel samples of pink-and red-fruited genotypes during ripening. Genetic mapping and segregation analysis showed that MYB12 is located on chromosome 1 and segregates perfectly with the characteristic pink fruit color. Virus-induced gene silencing of SlMYB12 resulted in a decrease in the accumulation of naringenin chalcone, a phenotype consistent with the pink-colored tomato fruit of IL1b. In conclusion, biochemical and molecular data, gene mapping, segregation analysis, and virus-induced gene silencing experiments demonstrate that the MYB12 transcription factor plays an important role in regulating the flavonoid pathway in tomato fruit and suggest strongly that SlMYB12 is a likely candidate for the y mutation.Plants produce pigments to provide color to their flowers and fruit for attracting pollinators and seed dispersers. In crop plants, color is regarded as one of the most important consumer traits in fruit such as tomato (Solanum lycopersicum), pepper (Capsicum annuum), apple (Malus domestica), and strawberry (Fragaria spp.). In flowers and fruit, these colors are due mainly to carotenoid and flavonoid pigments.Carotenoids are lipid-soluble 40-carbon isoprenoids, and more than 700 naturally occurring carotenoids have been identified (Britton et al., 2004). Carotenoids are essential for plant life, since they provide important photoprotective functions during photosynthesis in chloroplasts and serve as precursors for the phytohormone abscisic acid (Grotewold, 2006). Beyond their essential biological activities, carotenoids also accumulate in chromoplasts of flowers and fruit, where they function as yellowto red-colored pigments (Young and Frank, 1996;Tanaka et al., 2008). Carotenoids ...