Botulinum toxin is a zinc-dependent endoprotease that acts on vulnerable cells to cleave polypeptides that are essential for exocytosis. To exert this poisoning effect, the toxin must proceed through a complex sequence of events that involves binding, productive internalization, and intracellular expression of catalytic activity. Results presented in this study show that soluble chelators rapidly strip Zn 2؉ from its binding site in botulinum toxin, and this stripping of cation results in the loss of catalytic activity in cell-free or broken cell preparations. Stripped toxin is still active against intact neuromuscular junctions, presumably because internalized toxin binds cytosolic Zn 2؉ . In contrast to soluble chelators, immobilized chelators have no effect on bound Zn 2؉ , nor do they alter toxin activity. The latter finding is because of the fact that the spontaneous loss of Zn 2؉ from its coordination site in botulinum toxin is relatively slow. When exogenous Zn 2؉ is added to toxin that has been stripped by soluble chelators, the molecule rebinds cation and regains catalytic and neuromuscular blocking activity. Exogenous Zn 2؉ can restore toxin activity either when the toxin is free in solution on the cell exterior or when it has been internalized and is in the cytosol. The fact that stripped toxin can reach the cytosol means that the loss of bound Zn 2؉ does not produce conformational changes that block internalization. Similarly, the fact that stripped toxin in the cytosol can be reactivated by ambient Zn 2؉ or exogenous Zn 2؉ means that productive internalization does not produce conformational changes that block rebinding of cation.