Phosphotriesterase, isolated from the soil-dwelling bacterium Pseudomonas diminuta, catalyzes the detoxification of organophosphate-based insecticides and chemical warfare agents. The enzyme has attracted significant research attention in light of its possible employment as a bioremediation tool. As naturally isolated, the enzyme is dimeric. Each subunit contains a binuclear zinc center that is situated at the C-terminal portion of a "TIM" barrel motif. The two zincs are separated by ∼3.4 Å and coordinated to the protein via the side chains of His 55, His 57, His 201, His 230, Asp 301, and a carboxylated Lys 169. Both Lys 169 and a water molecule (or hydroxide ion) serve to bridge the two zinc ions together. Interestingly, these metals can be replaced with cadmium or manganese ions without loss of enzymatic activity. Here we describe the three-dimensional structures of the Zn2+/Zn2+-, Zn2+/Cd2+-, Cd2+/Cd2+-, and Mn2+/Mn2+-substituted forms of phosphotriesterase determined and refined to a nominal resolution of 1.3 Å. In each case, the more buried metal ion, referred to as the α-metal, is surrounded by ligands in a trigonal bipyramidal ligation sphere. For the more solvent-exposed or β-metal ion, however, the observed coordination spheres are either octahedral (in the Cd2+/Cd2+-, Mn2+/Mn2+-, and the mixed Zn2+/Cd2+-species) or trigonal bipyramidal (in the Zn2+/Zn2+-protein). By measuring the anomalous X-ray data from crystals of the Zn2+/Cd2+-species, it has been possible to determine that the α-metal ion is zinc and the β-site is occupied by cadmium.