The unique ability of a family of botulinum neurotoxins to block neuroexocytosis specifically-by selective interaction with peripheral cholinergic nerve endings, endocytotic uptake, translocation to the cytosol, and enzymic cleavage of essential proteins-underlies their increasing therapeutic applications. Although clinical use of type A is most widespread due to its prolonged inactivation of the synaptosomal-associated protein of 25 kDa, botulinum neurotoxin E cleaves this same target but at a different bond and exhibits faster onset of neuromuscular paralysis. Herein, insights were gained into the different dynamics of action of types A and E toxins, which could help in designing variants with new pharmacological profiles. Natural and recombinant type E dichain forms showed similar proteolytic and neuromuscular paralytic activities. The neuroparalysis induced by type E toxin was accelerated between 21 and 35 degrees C and attenuated by bafilomycin A1. Temperature elevation also revealed an unanticipated bipartite dose response indicative of two distinct internalization processes, one being independent of temperature and the other dependent. Although elevating the temperature also hastened intoxication by type A, a second uptake mechanism was not evident. Increasing the frequency of nerve stimulation raised the uptake of type E via both processes, but the enhanced trafficking through the temperature-dependent pathway was only seen at 35 degrees C. These novel observations reveal that two membrane retrieval mechanisms are operative at motor nerve terminals which type E toxin exploits to gain entry via an acidification-dependent step, whereas A uses only one.