The phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase (phe), a key enzyme involved in the biosynthesis of the aromatic amino acid phenylalanine, expressed by the Escherichia coli gene aroG, which catalyzes the condensation of D-erythrose 4-phosphate with phosphoenolpyruvate (PEP) to give DAH 7-P, was cloned into the expression vector pT7-7 for overexpression in E. coil. Purified enzyme from this expression system was used to demonstrate that DAH 7-P synthase (phe) also catalyses the aldol-type condensation of PEP with the 5-carbon analogues D-arabinose 5-phosphate, D-ribose 5-phosphate, and 2-deoxy-D-ribose 5-phosphate to yield 3-deoxy-D-manno-octulosonate 8-phosphate, 3-deoxy-D-altro-octulosonate 8-phosphate, and 3,5-dideoxy-D-gluco(manno)-octulosonate 8-phosphate, respectively, as determined by H-1 NMR and other standard analytical methods. The kinetic parameters, K-m and V-max, for these reactions were determined. The 3- and 6-carbon phosphorylated monosaccharides, D,L-glyceraldehyde 3-phosphate and D-glucose 6-phosphate, as well as the nonphosphorylated 5-carbon analogues D-arabinose 5-phosphate, D-ribose 5-phosphate, and 2-deoxy-D-ribose 5-phosphate were not substrates.