Abstract The expression of κ-bungarotoxin in Escherichia coli from a synthetic gene results in the production of multiple species of polypeptide. These include not only biologically active κ-bungarotoxin but also a variety of inactive species, which include inactive monomers as well as disulfide-linked polymeric species. Identification of these species and their separation from the biologically active recombinant toxin is necessary for the use of the toxin in physiological and biochemical studies. This has been accomplished by a combination of ion-exchange and reverse-phase chromatography which results in a homogeneous toxin preparation. The active material produced is sufficient for many types of biological studies and for mutagenesis experiments directed at determining the structure-function relationships of toxin interactions with the neuronal nicotinic acetylcholine receptor. In addition, the κ-bungarotoxin produced in this manner has the distinct advantage over venompurified κ-bungarotoxin of not being contaminated with other venom components which could potentially affect experimental observations.