Xanthophylls are oxygenated carotenoids that perform critical roles in plants. Beta-carotene hydroxylases (beta-hydroxylases) add hydroxyl groups to the beta-rings of carotenes and have been cloned from several bacteria and plants, including Arabidopsis. The lut1 mutation of Arabidopsis disrupts epsilon-ring hydroxylation and has been suggested to identify a related carotene hydroxylase that functions specifically on epsilon-ring structures. We have used library screening and genomics-based approaches to isolate a second beta-hydroxylase genomic clone and its corresponding cDNA from Arabidopsis. The encoded protein is 70% identical to the previously reported Arabidopsis beta-hydroxylase 1. Phylogenetic analysis indicates a common origin for the two proteins, however, their different chromosomal locations, intron positions and intron sizes suggest their duplication is not recent. Although both hydroxylases are expressed in all Arabidopsis tissues analyzed, beta-hydroxylase 1 mRNA is always present at higher levels. Both cDNAs encode proteins that efficiently hydroxylate the C-3 position of beta-ring containing carotenes and are only weakly active towards epsilon-ring containing carotenes. Neither beta-hydroxylase cDNA maps to the LUT1 locus, and the genomic region encompassing the LUT1 locus does not contain a third related hydroxylase. These data indicate that the LUT1 locus encodes a protein necessary for epsilon-ring hydroxylation but unrelated to beta-hydroxylases at the level of amino acid sequence.