The binding of sulfate, selenate and thiosulfate by the sulfur-transferase rhodanese (EC 2.8.1.1) in the crystalline state has been studied by X-ray analysis at resolutions between 0.23 nm and 0.4 nm. The three ions appear to occupy a common site between the N eta atoms of Arg-29 and the main-chain NH group of Glu-148 at the surface of the enzyme molecule. A second binding site for the three ions is situated at the entrance to the active centre, between the side chains of Arg-186 and Lys-249. Selenate and thiosulfate are bound equally well at both anion-binding sites. Sulfate, however, binds better at the first position, near Arg-29, than at the second site near Arg-186. In the complex of sulfur-rhodanese with thiosulfate, the outer sulfur atom of the anion near the active centre points towards the extra sulfur atom which is bound as a persulfide to the S gamma of the essential Cys-247. The distance between the outer sulfur atom of the thiosulfate ion and the persulfide sulfur atom appears to be about 0.3 nm. The thiosulfate difference Fourier also shows a distinct, localized conformational change involving residues 71, 72 and 249. This is the result of the replacement of an ammonium ion in the sulfate and selenate media by a sodium ion in the sodium thiosulfate solution. Rhodanese is apparently able to accomodate ions with different radii at this cation-binding site by minor structural alterations.