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Artemisia annua produces a large number of unique terpenoids, making it of particular interest as a source of phytochemicals and a useful model plant for studying terpenoid metabolism. The ability to engineer fast-growing in vitro cultures to produce terpenoids in high yield would be a dramatic step towards commercial use. Two distinct pathways have been characterized in higher plants leading to the biosynthesis of isopentenyl diphosphate, the common precursor to all terpenes: the cytosolic mevalonate pathway and the plastid-localized mevalonate-independent pathway. While transformed roots of A. annua have been demonstrated to be superior to whole plants in terms of yield of the sesquiterpene artemisinin, they appear to lack functional chloroplasts, bringing into question the presence of a functional mevalonate-independent pathway. Using a cDNA library made from these roots, we isolated two clones encoding deoxy-d-xylulose-5-phosphate synthase (DXPS) and deoxy-d-xylulose-5-phosphate reductoisomerase (DXPR). The biochemical function of both enzymes was confirmed by complementing E. coli dxps- and dxpr-mutants. Northern blot analysis showed that the transformed root cultures expressed these genes at different levels during the culture cycle. In addition, cultures grown in continuous light showed substantial increases in DXPS transcript levels compared to dark-grown cultures. These results represent an important step towards demonstrating the presence of the plastid-localized terpenoid biosynthetic pathway in these easily engineered in vitro cultures.