The recombinant, catalytically active light chain of botulinum toxin type A was evaluated as a potential vaccine candidate. Previous studies have shown that the light chain can elicit protective immunity in vivo. [Kiyatkin N, Maksymowych AB, Simpson LL. Induction of immune response by oral administration of recombinant botulinum toxin. Infect Immun 1997;65(11):4586-91], but the underlying basis for this observation was not determined. In the present study, antibodies directed against the light chain were shown to act at three different sites in the body to produce neutralization. Firstly, these antibodies acted to block toxin absorption into the body. This was demonstrated in vitro, in studies on binding and transport of toxin across epithelial monolayers, and in vivo, in studies on inhalation poisoning. Secondly, anti-light chain antibodies acted to promote clearance of toxin from the general circulation. This was demonstrated in vivo in studies on toxin levels in blood and in parallel studies on toxin accumulation in liver and spleen. Finally, anti-light chain antibodies acted to protect cholinergic nerves from botulinum toxin action. This was demonstrated in two types of in vitro assays: rate of paralysis of murine phrenic nerve-hemidiaphragm preparations and extent of binding to Neuro-2a cells. When taken together, these data show that anti-light chain antibodies can evoke three layers of protection against botulinum toxin.