Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis is an unusual enzyme because it contains an essential iron-sulfur center but does not catalyze an obvious oxidation-reduction reaction. In this communication, results of revised sulfide analyses, iron-sulfur cluster displacement studies, and Mössbauer spectroscopy are presented that lead to the conclusion that the native enzyme contains a tetranuclear [4Fe-4S] center in a diamagnetic state.