Lutein, a dihydroxy xanthophyll, is the most abundant carotenoid in plant photosynthetic tissues and plays crucial structural and functional roles in the light-harvesting complexes. Carotenoid beta-and epsilon-hydroxylases catalyze the formation of lutein from alpha-carotene (beta,epsilon-carotene). In contrast to the well studied beta-hydroxylases that have been cloned and characterized from many organisms, the epsilon-hydroxylase has only been genetically defined by the lut1 mutation in Arabidopsis. We have isolated the LUT1 gene by positional cloning and found that, in contrast to all known carotenoid hydroxylases, which are the nonheme diiron monooxygenases, LUT1 encodes a cytochrome p450-type monooxygenase, CYP97C1. Introduction of a null mutant allele of LUT1, lut1-3, into the beta-hydroxylase 1/beta-hydroxylase 2 (b1 b2) double-mutant background, in which both Arabidopsis beta-hydroxylases are disrupted, yielded a genotype (lut1-3 b1 b2) in which all three known carotenoid hydroxylase activities are eliminated. Surprisingly, hydroxylated beta-rings were still produced in lut1-3 b1 b2, suggesting that a fourth unknown carotenoid beta-hydroxylase exists in vivo that is structurally unrelated to beta-hydroxylase 1 or 2. A second chloroplast-targeted member of the CYP97 family, CYP97A3, is 49% identical to LUT1 and hypothesized as a likely candidate for this additional beta-ring hydroxylation activity. Overall, LUT1 defines a class of carotenoid hydroxylases that has evolved independently from and uses a different mechanism than nonheme diiron beta-hydroxylases.