Cholera toxin (CT) was shown to bind to immobilized Ni2+ ion. The affinity of CT for the complex required the presence of the Ni2+ ion, since CT was unable to bind in its absence. Binding was mediated by the B-subunit (CTB) as both CT and CTB bound to the resin, but not the A-subunit (CTA). Binding was reversible in the presence of imidazole and suggested that the affinity of CT for the Ni2+ ion was mediated by His residues. The heat-labile enterotoxin of Escherichia coli (LT), which is closely related to CT, was unable to bind to the Ni2+ ion. Comparison of amino acid sequences revealed the presence of three His residues in CT (positions 13, 57 and 94), but only one in LT (position 57). To confirm that the residues at positions 13 and 94 of CTB were responsible for the binding, they were changed to residues found in LTB. Changing His13-->Arg completely abrogated the ability of CTB to bind to Ni2+ ion. In contrast, the mutation of His 94-->Asn reduced, but did not abrogate, the ability of CTB to bind to Ni2+ ion. Based on calculated interatomic distances, it is unlikely that His13 and His94 are part of the same complex. There appear to be two separate binding sites, with the principal site involving His13 and a much weaker site involving His94. This latter site can only participate in binding if the complex involving His13 has formed.