The pathway of carotenoids starts with the synthesis of phytoene and proceeds along a single path up to lycopene which can be transformed to β-carotene by the action of lycopene β-cyclase or to α-carotene through the sequential action of lycopene ε-cyclase and lycopene β-cyclase. All xanthophylls are produced from these two cyclic precursors following two hydroxylation steps. β,β-Xanthophyll biosynthesis requires hydroxylases belonging to the so-called 'non-heme di-iron' group while the biosynthesis of lutein involves enzymes belonging to the vast group of P450 monooxygenases with different enzymatic specificity due to the distinct rings of α-carotene. Here we report on the isolation and functional characterization of tomato CYP97A29 and CYP97C11 genes encoding the P450 carotenoid β- and ε-hydroxylases. Through a reverse transcription-quantitative real-time PCR analysis of the two P450 and nine other carotenoid biosynthetic genes it was possible to highlight the transcriptional patterns of the 11 genes in root, leaf, petal and fruit at three stages of development and ripening. Finally, the characterization of the two P450 carotenoid (A29 and C11) hydroxylases was complemented by an in planta analysis through the use of transgenic plants. Results of this study have permitted us to model the lutein synthesis in leaf and in fruit of tomato.