The ability of chloroplasts to synthesize aromatic amino acids from CO2 was investigated using highly purified, intact spinach (Spinacia oleracea L. cv. Viking II) chloroplasts and 14CO2. Incorporation of 14C into aromatic amino acids was very low, however, and this was assumed to be due to lack of phosphoenolpyruvate (PEP), one of the substrates for the shikimate/arogenate pathway leading to aromatic amino acids in chloroplasts. Therefore, the glycolytic enzymes phosphoglycerate mutase (EC 18.104.22.168) and enolase (EC 22.214.171.124) were added to the 14CO2 fixation medium in order to convert labelled 3-phosphoglycerate exported from the intact chloroplasts to 2-phosphoglycerate and PEP. In this way a part of the glycolytic pathway was reconstituted outside the chloroplasts to substitute for the cytoplasm lost on isolation. The presence of both enzymes in the medium increased incorporation of 14C into Tyr and Phe more than ten-fold and incorporation into Trp about two-fold, while total 13CO2 fixation rates were not affected. Our results suggest that chloroplasts do not contain phosphoglycerate mutase or enolase, and that, in vivo, PEP is synthesized in the cytoplasm and imported to the chloroplast stroma for the biosynthesis of aromatic amino acids. The biosynthesis of all three aromatic amino acids was under feedback control. Using expected physiological concentrations (below 100 μM), each of the aromatic amino acids exerted a strict feedback inhibition of its own biosynthesis only.