The incorporation of [1-13 C]-and [2,3,4,5-13 C 4 ]1-deoxy-D-xylulose into -carotene, lutein, phytol, and sitosterol in a cell culture of Catharanthus roseus was analyzed by NMR spectroscopy. The labeling patterns of the isoprene precursors, isopentenyl pyrophosphate and dimethylallyl pyrophosphate, were obtained from the terpenes by a retrobiosynthetic approach. 13 C Enrichment and 13 C 13 C coupling patterns showed conclusively that 1-deoxy-D-xylulose and not mevalonate is the predominant isoprenoid precursor of phytol, -carotene, and lutein. Label from 1-deoxyxylulose was also diverted to phytosterols to a minor extent (6% relative to carotene and phytol formation). The data demonstrate that the formation of isopentenyl pyrophosphate from pentulose occurs strictly by an intramolecular rearrangement process.