Carotenoids are essential components of the photosynthetic apparatus in a wide range of organisms. They participate in the adaptation of plastids to changing environmental light conditions and prevent photo-oxidative damage of the photosynthetic apparatus by detoxifying reactive oxygen species. We identified eight cDNAs from the carotenoid biosynthetic pathway (PSY, PDS, ZDS, PTOX, LCY-E, CRTR-B, ZEP and VDE) and two cDNA encoding carotenoid cleavage dioxygenase family members (NCED3 and CCD1) in Coffea canephora. We also obtained cDNA encoding several different fibrillin proteins involved in carotenoid sequestration (FIB). Expression of the coffee carotenoid genes was determined in leaf, branch and flower tissues using quantitative RT-PCR. Expression analysis of these genes in leaf tissue from osmotically stressed plants was also carried out. These experiments showed that the transcript levels of PTOX, CRTR-B, NCED3, CCD1 and FIB1 increased under these stress conditions, while LCY-E decreased, indicating that the metabolic flux towards the xanthophyll cycle branch of the carotenoid biosynthetic pathway may be favoured in leaves under drought conditions. Functional analysis of CcCRTR-B using an in vivo method employing Escherichia coli strains engineered to make carotenoids confirmed that the beta-carotene hydroxylase activity of CcCRTR-B generates beta-cryptoxanthin and zeaxanthin from beta-carotene. A similar approach was also used to show that CcCCD1 encoded a functional 9,10(9'10') carotenoid cleavage dioxygenase, and thus that this enzyme is capable of forming one or more apocarotenoids in vivo. Finally, high-performance liquid chromatography analysis of coffee leaves revealed the presence of alpha-carotene and suggests that Coffea arabica may have higher levels of alpha-carotene than C. canephora.