Carotene hydroxylases catalyze the hydroxylation of a-and b-carotene hydrocarbons into xanthophylls. In red algae, b-carotene is a ubiquitously distributed carotenoid, and hydroxylated carotenoids such as zeaxanthin and lutein are also found. However, no enzyme with carotene hydroxy-lase activity had been previously identified in red algae. Here, we report the isolation of a gene encoding a cytochrome P450-type carotene hydroxylase (PuCHY1) from Porphyra umbilicalis, a red alga with an ancient origin. Sequence comparisons found PuCHY1 belongs to the CYP97B subfamily, which has members from different photosynthetic organisms ranging from red algae to land plants. Functional comple-mentation in Escherichia coli suggested that PuCHY1 catalyzed the conversion from b-carotene to zeaxanthin. When we overexpressed PuCHY1 in the Arabidopsis thaliana chy2 mutant, pigment analysis showed a significant accumu-lation of hydroxylated carotenoids, including neoxanthin,violaxanthin, and lutein in the leaves of transgenic plants. These results confirmed a b-hydroxylation activity of PuCHY1, and also suggested a possible e-hydroxylation function. The pigment profile and gene expression analyses of the algal thallus under high-light stress suggested that P. umbilicalis is unlikely to operate a partial xanthophyll cycle for photoprotection.