Iron K-edge X-ray absorption data on the purple acid phosphatase from porcine uterus (uteroferrin, Uf) have been obtained for the native reduced enzyme and for the oxidized enzyme in its phosphate- and arsenate-bound forms. In all three complexes, the first sphere consists of 1.5N/O at ~ 1.94 Å, 4 N/O at ~2.1 Å, and 0.5-1 N/O at ~2.4 Å; in no complex is found an Fe-O bond of ~1.8 Å which would derive from a µ-oxo bond. The ~1.94-Å shell corresponds to Fe-OAr and Fe-µ-OH(or R) bonds. The ~2.1-Å shell arises from histidine, carboxylate, oxoanion, and solvent ligation. The scatterer at ~2.4 Å is associated with a chelated carboxylate residue. The second-sphere analysis for Ufr indicates an Fe-Fe distance of 3.52 Å, similar to those found for semimethemerythrin azide, methane monooxygenase, and related model complexes, which suggests the presence of a (µ-hydroxo or alkoxo)diiron unit supported by a carboxylate bridge. On the basis of the EXAFS analysis and other spectroscopic data, it is proposed that tyrosine and histidine are terminal ligands to the Fe(III) center, and histidine and the chelated carboxylate coordinate to the Fe(II) center, with solvent molecules completing the diiron coordination sphere. This proposed active site is slightly modified from that found for the R2 protein of ribonucleotide reductase from Escherichia coli and suggested for the hydroxylase component of methane monooxygenase. The diiron cores of Ufo-PO4 and Ufo·AsO4 are significantly different from that of Ufr. Given the absence of a 1.8-Å bond, the diferric sites are not oxo-bridged, a conclusion also corroborated by the small intensity of the is → 3d preedge features in these complexes. The Fe-Fe distances of ~ 3.2–3.3 Å found for Ufo·PO4 and Ufo·AsO4 must then arise from an Fe2(OR)2 core. The observed Fe-P (3.17 Å) and Fe-As (3.41 Å) distances correspond to Fe-O-P(As) angles indicative of a bidentate bridging oxoanion which supports the Fe2O2 core.