To study regulation of the plastid-localized maize carotenoid biosynthetic pathway, a cDNA encoding phytoene desaturase (PDS) was isolated and characterized. The DNA sequence of the maize Pds cDNA was determined and compared with available dicot Pds genes. The deduced PDS protein, estimated at 64.1 kDa (unprocessed), had a dinucleotide binding domain and conserved regions characteristic of other carotene desaturases. Alignment of available PDS sequences from distantly related organisms suggests that Pds has potential as a phylogenetic tool. By use of heterologous complementation in Escherichia coli, maize PDS was shown to catalyze two desaturation steps converting phytoene to zeta-carotene. RFLP (restriction fragment length polymorphism) mapping was used to place Pds on chromosome 1S near viviparous5 (vp5), and RT-PCR (reverse-transcriptase polymerase chain reaction) analysis indicated reduced Pds transcript in vp5 mutant relative to normal endosperm. Other phytoene-accumulating mutant endosperms, vp2 and white3 (w3), showed no difference in Pds transcript accumulation as compared with normal endosperm counterparts. RT-PCR analysis of Pds transcript accumulation in developing endosperm showed Pds was constitutively expressed. Therefore, endosperm carotenogensis is not regulated by increasing the level of Pds transcripts.