Tetanus toxin contains 14 histidine residues: six of them are localized in the light chain (L), one is present in the N-terminal half of the heavy chain (HN) and the remaining seven histidines are localized in the C-terminal half of the heavy chain (Hc). Using immobilized-metal-ion affinity chromatography with Chelating Superose-Zn(II), we show that histidines of Hc are exposed to the protein surface and are responsible for the binding of tetanus toxin and of Hc to the immobilized metal. The histidines of the L chain are not available for co-ordination of matrix-bound Zn2+; however, two of them and three of the histidines of fragment Hc are accessible to diethyl pyrocarbonate. Chromatography on Superose-Zn(II) is also shown to be a simple and efficient method for the rapid isolation of tetanus toxin and of its Hc fragment, which can be extended to the botulinum neurotoxins.