Abstract Human glutamine phosphoribosylpyrophosphate amidotransferase (PP-ribose-P amidotransferase; EC 2.4.2.14) has been purified 40-fold from placenta. The enzyme is located in the cytosol. The partially purified preparation is stable and free of glutaminase and nucleotidase activity, permitting study of the substrate kinetics and regulatory properties. Michaelis-Menten substrate kinetics were observed with apparent Km values of 0.48 mm for PP-ribose-P and 1.6 mm for glutamine. Human PP-ribose-P amidotransferase has an absolute requirement for a divalent cation, with the order of relative effectiveness Mg2+ = Mn2+ g Co2+ g Ca2+. Other divalent cations tested did not support catalytic activity. Orthophosphate has complex effects upon human PP-ribose-P amidotransferase; these include enzyme stabilization, competitive inhibition with PP-ribose-P and sensitization to nucleotide feedback inhibition. Human PP-ribose-P amidotransferase was inhibited by a wide variety of purine and pyrimidine nucleotides; the corresponding nucleosides and bases were ineffective. Inhibitory effectiveness decreased with the series mono- g di- g triphosphonucleotide. Certain preparations of PP-ribose-P amidotransferase lost the property of feedback inhibition, yet retained catalytic activity suggesting that the catalytic and regulatory sites were topologically distinct. Kinetic studies revealed that AMP produced a qualitative change in PP-ribose-P substrate curves, from hyperbolic to sigmoidal, with an attendent change in Hill coefficient from 1.1 to 2.7 at 3.0 mm AMP. AMP was a noncompetitive inhibitor with respect to glutamine and AMP did not qualitatively alter glutamine substrate curves. Combinations of 6-OH- and 6-NH2-purine nucleotides resulted in synergistic inhibition. This suggests separate but interacting binding sites for these end products. The pyrimidine nucleotides methyl-dCMP and TMP were also found to exhibit synergistic inhibition with AMP. The kinetic and regulatory properties of human PP-ribose-P amidotransferase are in accord with a postulated role for this enzyme in the in vivo regulation of prime biosynthesis de novo.