In ripening tomato fruits, the leaf-specific carotenoids biosynthesis mediated by phytoene synthase 2 (PSY2) is replaced by a fruit-specific pathway by the expression of two chromoplast-specific genes: phytoene synthase 1 (PSY1) and lycopene-β-cyclase (CYCB). Consequently, mutations in those two and other genes contributing to intermediate steps render the ripened tomato fruits bereft of lycopene. To decipher whether PSY2-mediated pathway also operates in ripening fruits, we blocked the in vivo activity of lycopene-β-cyclases by injecting CPTA (2-(4-Chlorophenylthio) triethylamine hydrochloride), an inhibitor of lycopene-β-cyclases. The injection of CPTA induced accumulation of lycopene in leaves, immature-green and ripening fruits. Even, in tomato mutants deficient in fruit-specific carotenoid biosynthesis such as V7 and r (PSY1), and ζ-carotene isomerase (ZISO), CPTA triggered lycopene accumulation. The CPTA-treated ziso mutant fruits, where PSY1 remains functional, accumulated phytoene and phytofluene. Conversely, CPTA-treated PSY1-knockout mutant (r3756) fruits did not accumulate phytoene and phytofluene. CPTA-treated fruits were enriched in lycopene-derived volatiles and had reduced ABA levels. The lycopene accumulation was associated with the partial transformation of chloroplasts to chromoplasts bearing thread-like crystalline structures, indicating lycopene accumulation. Our study shows that inhibition of lycopene β-cyclases unmasks the operation of a parallel carotenoid biosynthetic pathway mediated by PSY2 in ripening tomato fruits.