A procedure for the rapid and efficient purification of glutamine phosphoribosylpyrophosphate amidotransferase to better than 98% homogeneity from derepressed Bacillus subtilis cells is described. The molecular weight of the subunit was estimated to be about 50 000. The purified enzyme exhibits microheterogeneity on electrophoresis on highly resolving polyacrylamide gels; it is suggested that this heterogeneity results from limited proteolytic modification of the native subunit. The native enzyme exists in equilibrium among tetrameric, dimeric, and monomeric forms. The influence of enzyme concentration and the presence of substrates and allosteric inhibitors on this equilibrium are described. There is no simple correlation between allosteric inhibition and stabilization of dimeric or tetrameric states. The amino acid composition of the amidotransferase is reported; presence of a 4Fe-4S center in the enzyme was described previously. Preparation of inactive apoprotein by treatment with 1,10- phenanthroline and general characteristics of the apoprotein are presented.