Carotenoids are orange, yellow, or red photo-protective pigments present in all plastids. The first carotenoid of the pathway is phytoene, a colorless compound that is converted into colored carotenoids through a series of desaturation reactions. Genes coding for carotenoid desaturases have been cloned from microbes but not from plants. We report the cloning of a cDNA for pds1, a soybean (Glycine max) gene that, based on a complementation assay using the photosynthetic bacterium Rhodobacter capsulatus, codes for an enzyme that catalyzes the two desaturation reactions that convert phytoene into zeta-carotene, a yellow carotenoid. The 2281-base-pair cDNA clone analyzed contains an open reading frame with the capacity to code for a 572-residue protein of predicted Mr 63,851. Alignment of the deduced Pds1 peptide sequence with the sequences of fungal and bacterial carotenoid desaturases revealed conservation of several amino acid residues, including a dinucleotide-binding motif that could mediate binding to FAD. The Pds1 protein is synthesized in vitro as a precursor that, upon import into isolated chloroplasts, is processed to a smaller mature form. Hybridization of the pds1 cDNA to genomic blots indicated that this gene is a member of a low-copy-number gene family. One of these loci was genetically mapped using restriction fragment length polymorphisms between Glycine max and Glycine soja. We conclude that pds1 is a nuclear gene encoding a phytoene desaturase enzyme that, as its microbial counterparts, contains sequence motifs characteristic of flavoproteins.