Highly purified iodinated tetanus toxin preparations separate on ganglioside affinity columns into two distinct (A and B) fractions representing about 20% and 75% of the iodinated toxin, respectively. Fraction A, eluted by 1% NaCl, migrates like native tetanus toxin (150,000 mol. wt) on SDS polyacrylamide gel electrophoresis. It forms an aggregate of molecular weight approximately 360,000 on Sephacryl S-300 gel permeation chromatography in the presence of detergent and contains two isoforms on preparative chromatofocusing. Fraction A binds poorly to neurons in tissue culture or to synaptosomal membrane preparations. It retains, however, its antigenicity and biotoxicity. Fraction B, eluted by 6% NaCl, binds effectively to gangliosides and also to neurons or synaptosome preparations. It has a similar molecular weight and chain composition to the native toxin and displays two isoforms, precipitable during chromatofocusing. Fraction B possesses similar binding, immunological and toxic properties to the original iodinated tetanus toxin. Following excessive iodination (4-6 mCi/mg protein), toxicity can be remarkably reduced. Unlabeled toxin shows a similar chromatographic pattern to the iodinated toxin on affinity columns, suggesting that a large portion (30% by protein and 55% by toxicity) of the toxin has a poor affinity for gangliosides. The molecular pharmacokinetics of tetanus toxin with respect to affinity toward ganglioside-dependent and ganglioside-independent receptors needs re-evaluation.