Cell cultures of the plant Rubia tinctorum were grown with supplements of [1-C-13]- or [U-C-13(6)]-glucose. Amino acids were obtained by hydrolysis of biomass, and their C-13 labeling patterns were used to reconstruct the labeling patterns of acetyl CoA, pyruvate, phosphoenol pyruvate, erythrose 4-phosphate, and cl-ketoglutarate by retrobiosynthetic analysis. These patterns were used to construct hypothetical labeling patterns for the anthraquinone, lucidin primveroside, via different hypothetical pathways. The predicted labeling pattern based on the precursors o-succinylbenzoate and dimethylallyl pyrophosphate derived via the deoxyxylulose pathway was in excellent agreement with the observed labeling pattern of the anthraquinone derivative. The data show that the retrobiosynthetic concept can be used to quantitatively estimate the flux of metabolites via different metabolic pathways.