Tetanus toxin labeled by the Bolton-Hunter technique possesses high specific activity and retains substantial biological activity. This material can be used to characterize tetanus toxin binding to receptors in brain membrane preparations. In experiments aimed at measuring the absorption of labeled toxin, the displacement of labeled toxin by unlabeled toxin and the on-rate and off-rate constants, the data revealed two binding sites. The high affinity site had a Kd of 0.033 to 0.070 nM and a Bmax of 0.26 to 0.4 pmol/mg of protein; the low affinity site had a Kd of 0.89 to 6.9 nM and a Bmax of 1.55 to 3.0 pmol/mg of protein. The binding of tetanus toxin to brain membranes was enhanced greatly by low pH and ionic strength. Similarly to tetanus toxin, botulinum neurotoxin could be labeled by the Bolton-Hunter technique, and its binding to brain membranes was also enhanced by low pH and ionic strength. In studies with a neutralizing monoclonal antibody against tetanus toxin, the antigen-antibody interaction was not significantly altered by media with low ionic strength and pH. On the other hand, the ability of the antibody to block toxin binding to brain membranes was reduced substantially in nonphysiologic media. In a bioassay aimed at determining the effect of pH and tonicity on tissue association by toxin, low pH and ionic strength did not enhance toxicity. The biological activity of tetanus toxin was unaffected and that of botulinum neurotoxin was greatly diminished. The present findings confirm the widely reported observation that low pH and ionic strength promote tissue association by tetanus toxin, but they challenge the premise that this binding is relevant to the normal process of cell poisoning.