The regulation of aromatic amino acid biosynthesis in Nocardia sp. 239 was studied. In cell-free extracts 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase activity was inhibited in a cumulative manner by tryptophan, phenylalanine and tyrosine. Chorismate mutase was inhibited by both phenylalanine and tyrosine, whereas prephenate dehydratase was very sensitive to inhibition by phenylalanine. Tyrosine was a strong activator of the latter enzyme, whereas anthranilate synthase was inhibited effectively by tryptophan. No clear repression of the synthesis of these enzymes was observed during growth of the organism in the presence of the aromatic amino acids. It is therefore concluded that in Nocardia sp. 239 synthesis of these amino acids is mainly regulated by feedback inhibition. The molecular organization and kinetic properties of DAHP synthase were studied in more detail following its purification. The molecular weight of the native enzyme and its single subunit species were estimated to be 168,000 and 41,000, respectively, suggesting that the enzyme is a tetramer. Apparent Km values for phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) were 45 and 370 gM, respectively. Tryptophan, phenylalanine and tyrosine inhibited DAHP synthase in a competitive manner with respect to E4P, with apparent Ki values of 3, 160 and 180 gM, respectively. In addition, tryptophan and E4P (apparent Ki values of 11 and 530 gM, respectively) were found to exert an uncompetitive and competitive inhibition, respectively, towards PEP.