Visual pigments (rhodopsins) are composed of a chromophore (vitamin A derivative) bound to a protein moiety embedded in the retinal membranes. Animals cannot synthesize the visual chromophore de novo but rely on the uptake of carotenoids, from which vitamin A is formed enzymatically by oxidative cleavage. Despite its importance, the enzyme catalyzing the key step in vitamin A formation resisted molecular analyses until recently, when the successful cloning of a cDNA encoding an enzyme with β,β-carotene-15,15′-dioxygenase activity from Drosophila was reported. To prove its identity with the key enzyme for vitamin A formation in vivo , we analyzed the blind Drosophila mutant ninaB . In two independent ninaB alleles, we found mutations in the gene encoding the β,β-carotene-15,15′-dioxygenase. These mutations lead to a defect in vitamin A formation and are responsible for blindness of these flies.