Two proteins essential for the biosynthesis of the amino acid histidine in Escherichia coli have been overexpressed and purified to apparent homogeneity. The protein encoded by the hisF gene has an ammonia-dependent activity that results in the conversion of the biosynthetic intermediate N1-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4- carboxamide ribonucleotide (PRFAR) to imidazole glycerol phosphate (IGP) and 5-aminoimidazole-4-carboxamido-1-beta-D- ribofuranosyl 5'-monophosphate (AICAR). The second protein encoded by the hisH gene exhibits no detectable catalytic properties with biosynthetic intermediate PRFAR, glutamine, or ammonia. In combination, the proteins are capable of a stoichiometric conversion of glutamine and PRFAR to form AICAR, IGP, and glutamate. Neither protein alone is capable of mediating a conversion of the nucleotide substrate to a free metabolic intermediate. The HisH and HisF proteins form a stable 1:1 dimeric complex that constitutes the IGP synthase holoenzyme. Steady-state kinetic parameters for the holoenzyme indicate that glutamine is a more efficient substrate relative to ammonium ion by a factor of 10(3). The HisF subunit will support an ammonia-dependent reaction with a turnover number similar to that of the holoenzyme with glutamine. The glutaminase activity for the holoenzyme is 0.8% of that in the presence of the nucleotide substrate PRFAR. There are critical subunit interactions that mediate the catalytic properties for glutamine hydrolysis. The catalytic turnover of glutamine can be increased up to 37-fold by the addition of either the product IGP or the biosynthetic precursor N1-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (5'-ProFAR). The mechanistic significance of this glutaminase activity compared to other trpG type glutamine amidotransferases is discussed.