The ascomycete Fusarium fujikuroi produces carotenoids by means of the enzymes encoded by three car genes. The enzymes encoded by carRA and carB are responsible of the synthesis of beta-carotene and torulene, respectively, while the product encoded by carT cleaves torulene to produce the acidic xanthophyll neurosporaxanthin. carRA and carB are found in a cluster with a third gene, carO, which codes for an opsin-like protein. However, no information is available on the sequence or chromosomal location of carT, which has been identified only by mutant analysis. Transcription of the three clustered genes is stimulated by light and by mutations in a regulatory gene, leading to overproduction of carotenoids. We have now identified a fourth gene in the car cluster, called carX, which codes for a protein similar to known carotenoid-cleaving oxygenases. carX is transcribed divergently from carRA, and exhibits the same transcriptional pattern as carRA, carB and carO. Targeted deletion of carX resulted in a phenotype characterized by a significant increase in the overall carotenoid content. In the dark, the carX mutants accumulate at least five times more carotenoids than the wild type, and exhibit partial derepression of carRA and carB transcription. The mutants also show more intense pigmentation in the light, but the increase in the carotenoid content relative to the wild type is less than twofold. Under these conditions, the mutants also show a relative increase in the amounts of phytoene and cyclic carotenoids formed, suggesting that CarRA activity is enhanced.