Uteroferrin (Uf) is the purple acid phosphatase from porcine uterus and contains a coupled dinuclear iron unit. The reduced form [Fe(III), Fe(II)J is pink (λmax = 510 nm), is catalytically active toward phosphate ester hydrolysis, and has EPR signals (gav = 1.74) characteristic of an S = 1/2 ground state arising from a mixed-valence metal site. The Fe(II) site can be replaced with Zn(II) resulting in an Fe(III), Zn(II) form which is catalytically active, retains a purple color (λmax = 530 nm), and has EPR signals characteristic of high-spin Fe(III). Anions such as phosphate, arsenate, and molybdate bind to FeZnUf and Ufr with similar affinities, resulting in the inhibition of phosphate ester hydrolysis. The spectroscopic changes elicited upon binding of these anions to either Ufr or FeZnUf afford new insights into the coordination chemistry of the dinuclear site. EPR experiments with 17O-labeled phosphate provide the first evidence for direct coordination of phosphate to this novel dinuclear metal active site, while the effects on H2O/D2O substitution of the EPR spectrum of UfrAsO4 support a model wherein binding of phosphate and arsenate entails at least some proton transfer from the anion to the hydroxo bridge. These results, when extrapolated to the enzyme-substrate interaction, suggest a novel role for the dinuclear unit in the hydrolysis of phosphate esters.