Neuronal-growth-inhibitory factor (GIF) is a metalloprotein specific to the central nervous system which has been linked to Alzheimer's disease. The high metal content, approximately seven metal atoms/protein molecule, and 70% sequence identity to mammalian metallothioneins (MT), including a preserved array of 20 cysteinyl residues, place GIF in the family of MT. In contrast to MT, native GIF isolated from human or bovine brain contains an unusual metal composition, viz. four Cu(I) and three Zn(II) per polypeptide chain. Cu and/or Zn K-edge X-ray absorption spectra have been recorded for native Cu, Zn-GIF, Zn-substituted GIF, and these metals bound to the 32-residue N-terminal domain, Cu4-, Cu6- or Zn3-GIF-(1-32) at 77 K. The results are consistent with the metals being bound to the protein by cysteinyl residues in every case. The Cu-S distance is approximately 2.25 A and the EXAFS is considered to be consistent with primarily trigonal coordination of the Cu(I); Cu...Cu backscattering is observed at approximately 2.67 A, indicative of the formation of Cu(x)(Scys)y clusters. Thus, the Cu(I) environment is similar to that observed in MT. This is also the case for Zn(II), with 4 S at approximately 2.34 A. However, in contrast to Zn-MT for Zn-substituted GIF and Zn3-GIF-(1-32), Zn...Zn backscattering is observed at approximately 3.28 A. The significance of these results are discussed with respect to the specific biological activity of GIF.