Inhibition of biosynthetic enzymes and translation and translocation processes, elicitation, and precursor feeding were used to probe biosynthetic pathway compartmentation, substrate-product relationships, and yield limitation of the diterpenoid taxanes in cell cultures of Taxus chinensis (PRO1-95). The results suggest the following : (i) the source of isopentenyl pyrophosphate in taxane production is likely plastidic rather than cytoplasmic; (ii) baccatin III may not be a direct precusor of Taxol (Taxol is a registered trademark of Bristol-Myers Squibb for paclitaxel); (iii) baccatin III appears to have cytoplasmic and plastidic biosynthetic components, while Taxol production is essentially plastidic; and (iv) arachidonic acid specifically stimulates Taxol production but does not have a significant effect on baccatin III yield. Semiempirical mathematical models were used to describe these results and predict potential yield-limiting steps. Model simulations suggest that, under current operating conditions, Taxol production in Taxus chinensis (PRO1-95) cultures is limited by the ability of the cells to convert phenylalanine to phenylisoserine rather than by the branch-point acyl transferase. This result is supported by the lack of improvement of Taxol yield by feeding phenylalanine or benzoylglycine. The methods described in this article, while specifically expanding our knowledge of taxane production in PRO1-95 cultures, could be generally useful in investigating complex aspects of secondary metabolic pathways in plant cell cultures, especially when details of the pathway and compartmentation are sparse.