The formation of the powerful Zn binding protein called Zn-thionein was examined using male albino rats and [14C]cysteine, as cystein is known to be the most abundant constituent of this metal protein. 65% of the hepatic [14C]cysteine was incorporated into the protein portion of freshly prepared Zn-thionein. The protein was isolated by a combination of ethanol/chloroform treatment and various chromatographic steps, including ion exchange and gel filtration. 4.7 mol of Zn, 0.02 mol of Cd and less than 0.001 mol of either Cu or Hg were found per 12 000 g of portein. It was presumed that considerable amounts of Zn were lost during these isolation procedures, with the consequence of disulphide gridge formation. Indeed, the presence of R-S-S-R was deduced from circular dichroism and X-ray photoelectron spectroscopy. Due to the clearly detectable disulphide chromophore in the circular dichroism spectrum, it was possible to assign the shoulder at S 2p1/2,3/2 = 162.7 eV of the X-ray photoelectron spectrum of native Zn-thionein to R-S-S-R and not to strongly polarized sulphur. Upon reducing R-S-S-R-containing native Zn-thionein with dithiothreitol, all oxidised thiolate moieties of the thionein molecule could be restored. The addition of ZnCl2 with the subsequent desalting of extraneously bound Zn2 yielded a homogeneous Zn-thionein with 9.6 mol Zn2 per mol protein. A stoichiometry of ZnRS 1:3 was seen, which confirmed earlier reports of the existence of the mixed Cd,Zn-thionein. The conversion of mixed Cd,Zn-thionein into homogeneous Zn-, Cd-, Hg- and Cu-thionein by the gel filtration technique proved successful. From chiroptical measurements, the extraordinary contribution of the metal chromophores to the circular dichroism was seen. Due to the differences in the geometry of complexes formed by the respective metal ions, dramatic changes in the protein portion were expected. Polyacrylamide disc electrophoresis of purified native untreated Zn-thionein resulted in the appearance of two or more bands. This phenomenon was attributed to the different migration rates of cystine-thionein and thiolate-rich Zn-thionein, and was consistent with the spectral properties of the above Zn-protein species. By contrast, only one single band was monitored when a homogeneous metal-thionein was electrophoresed.