Xanthophyllomyces dendrorhous is one of the relevant sources of the carotenoid astaxanthin. In this paper, we describe for the first time cloning of unexpected cDNAs obtained from the crtI and crtYB genes of X. dendrorhous strain UCD 67-385. The cDNA of the crtI gene conserves 80 bp of the first intron, while the cDNA of the crtYB gene conserves 55 bp of the first intron and lacks 111 bp of the second exon. The crtI and crtYB RNAs could be spliced in alternative splice sites, which produced alternative transcripts which could not be translated to active CRTI and CRTYB proteins since they had numerous stop codons in their sequences. The ratio of mature mRNA to alternative mRNA for the crtI gene decreased as a function of the age of the culture, while the cellular content of carotenoids increased. It is possible that splicing to mature or alternative transcripts could regulate the cellular concentrations of phytoene desaturase and phytoene synthase-lycopene cyclase proteins, depending on the physiological or environmental conditions.Carotenoids are terpenoid pigments that are synthesized in bacteria, algae, fungi, and green plants (5). They provide protection against photooxidation and inactivate free radicals due to their highly conjugated double-bond systems (11,28,29). Astaxanthin, produced primarily by phytoplankton, is the principal carotenoid responsible for the orange-red color of marine invertebrates, fish, and birds. Since animals are not capable of astaxanthin synthesis, this xanthophyll must be added to the feed of aquacultured organisms like salmonids to obtain the appropriate pigmentation for consumer appeal (19,38). To date, one of the few microbial sources of astaxanthin is the red basidiomycetous yeast Xanthophyllomyces dendrorhous (3,20,24).As in other organisms, the condensation of two molecules of geranylgeranyl pyrophosphate (GGPP) to phytoene is the first specific step in the biosynthesis of astaxanthin. Subsequently, the pathway to lycopene involves the introduction of double bonds by four desaturations of phytoene. Finally, after cyclization of lycopene to -carotene, hydroxylation occurs at the 3 and 3Ј carbons, and keto groups are added at positions 4 and 4Ј of -rings. Recently, the phytoene desaturase-encoding gene (crtI) was isolated by heterologous complementation in an Escherichia coli strain accumulating phytoene. To do this, a cDNA library from X. dendrorhous was introduced into an E. coli strain carrying genes encoding GGPP synthase (crtE) and phytoene synthase (crtB) from Erwinia uredovora (40). By using a similar approach, the first lycopene cyclase gene (crtYB) described in a fungus was isolated from X. dendrorhous (41). The product of the crtYB gene is a bifunctional enzyme with two catalytic activities, as it converts GGPP into phytoene and lycopene into -carotene. A related phytoene synthase-lycopene cyclase has been cloned from other fungi belonging to the taxonomic groups ascomycota and zygomycota (6, 7, 39).Additionally, numerous studies on the expression at the mRNA leve...