Deinococcus radiodurans strain R1 synthesizes a unique ketocarotenoid product named deinoxanthin. The detailed steps involved in the biosynthesis of deinoxanthin remain unresolved. A carotene ketolase homologue encoded by dr0093 was inactivated by gene mutation to verify its function in the native host D. radiodurans. Analysis of the carotenoids in the resultant mutant R1DeltacrtO demonstrated that dr0093 encodes gamma-carotene ketolase (CrtO) catalysing the introduction of one keto group into the C-4 position of gamma-carotene derivatives to form ketolated carotenoids. The mutant R1DeltacrtO became more sensitive to H(2)O(2) treatment than the wild-type strain R1, indicating that the C-4 keto group is important for the antioxidant activity of carotenoids in D. radiodurans. Carotenoid extracts from mutant R1DeltacrtO exhibited lower 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity than those from the wild-type strain R1. The enhanced antioxidant ability of ketocarotenoids in D. radiodurans might be attributed to its extended conjugated double bonds and relative stability by the C-4 keto group substitution.