The stepwise 57Fe(II)-thiolate cluster formation in rabbit liver metallothionein-2 (MT) has been followed at pH 8.5 using Mössbauer spectroscopy. The zero-field spectra recorded at 4.2 K exhibit at all stages of filling one virtually identical single quadrupole splitting delta EQ and isomer shift delta as found for reduced rubredoxin (Rdred) or the model compound [Fe(II)(SPh)4]2-, thus indicating an Fe(II)-tetrathiolate coordination. A similar conclusion was reached also in previous electronic absorption studies [M. Good and M. Vasák (1986) Biochemistry 25,8353--8356]. The Mössbauer spectra obtained in the presence of a magnetic field were analyzed on the basis of a spin-Hamiltonian formalism resulting in Mössbauer parameters similar to those for Rdred and the inorganic model compound [Fe(II)(SPh)4]2-. The identity of the Mössbauer parameters of partially and fully metal-occupied MT suggests that a comparable distortion of the metal binding sites must exist. Simulation of the spectra revealed that the Fe(II) ions in the partially metal-occupied 57Fe(II)4-MT form appear to be magnetically isolated, whereas in the fully metal-saturated 57Fe(II)7-MT form a ratio of 3:4 of paramagnetic to diamagnetic subspectra was obtained. The latter result suggests the existence of three isolated metal binding sites and a metal-thiolate cluster containing four metal ions. In the light of structure determinations of MT containing Zn(II) and/or Cd(II) [W. Braun et al. (1986) J. Mol. Biol. 187, 125-129, and W. F. Furrey et al. (1986) Science (Wash. DC) 231, 704-710], which revealed two metal-thiolate clusters containing three and four metal ions, respectively, and involving all 20 cysteine residues in metal binding, the appearance of Mössbauer parameters characteristic of three isolated Fe(II) sites in 57Fe(II)7-MT is peculiar and deserves further studies. It is concluded, moreover, that the four-metal cluster is diamagnetic with the four Fe(II) ions being antiferromagnetically coupled. The appearance of magnetic coupling above four Fe(II) equivalents bound to apoMT indicates that the cluster formation occurs in a two-step process.