Carotenoids are plant secondary metabolites that comprise two main groups: carotenes and xanthophylls. The latter group includes zeaxanthin which is synthesized by beta-carotene hydroxylase catalyzing the hydroxylation of the beta-rings of beta-carotene molecules. To develop tools to alter carotenoid biosynthesis in plants, we isolated a cDNA clone encoding a candidate beta-carotene hydroxylase, CrtH1, from the flower petals of Adonis aestivalis. CrtH1 protein has homology to beta-carotene hydroxylases from other organisms, and possesses the four histidine motifs conserved in this family of enzymes. Sequence analysis predicted the presence of a putative plastid transit peptide at the amino terminus and four transmembrane helical regions. Southern-blot analysis showed CrtH1 to be encoded by a multicopy gene family with at least three members in A. aestivalis. Analysis of CrtH1 transcript abundance by Northern blotting indicates it is highly expressed in flower petals, roots and stems, with relatively low expression in leaves and developing seeds. CrtH1 was able to catalyze the formation of zeaxanthin and its intermediate precursor beta-cryptoxanthin from beta-carotene in functional assays conducted in E. coli. Expression of CrtH1 in Arabidopsis thaliana wild type and a mutant deficient for endogenous beta-carotene hydroxylases enhanced the biosynthesis of violaxanthin in the seeds.