In the purple photosynthetic bacterium Rhodobacter sphaeroides, the desaturation of phytoene has already been implicated in the assembly of the light-harvesting 2 complex (H.P. Lang and C.N. Hunter, Biochem. J. 298:197-205, 1994). The phytoene synthase and desaturase enzymes mediate the first steps specific for carotenoid biosynthesis up to and including the synthesis of the colored carotenoid neurosporene. In this report, we present the DNA and deduced amino acid sequences of the genes encoding these proteins, namely, crtB and crtI, from R. sphaeroides and present evidence for the existence of a crtIB operon. Both genes have been shown to possess putative puc and puf operon-like promoter sequences, and oxygen regulation and the point of initiation of the crtI transcript have been demonstrated. The complete crtI gene has been overexpressed in Escherichia coli and R. sphaeroides and shown to catalyze three desaturations of phytoene to give neurosporene. This activity was shown to be ATP dependent, and the cofactor requirement was investigated by using a spectroscopic assay for in vitro carotenogenic activity. Although the crtI and crtB genes have been sequenced from a number of different organisms, the transcriptional organization and regulation of these genes have not been analyzed in detail. In this report, we have located the transcription initiation point and have shown that R. sphaeroides possesses an oxygen-regulated CrtI-type phytoene desaturase gene that forms a transcriptional operon with crtB.