A major unmet clinical need exists for long-acting neurotherapeutics to alleviate chronic pain in patients unresponsive to available nonaddictive analgesics. Herein, a new strategy is described for the development of potent and specific inhibitors of the neuronal exocytosis of transmitters and pain mediators that exhibit unique antinociceptive activity. This entailed recombinant production in Escherichia coli of two serotypes of botulinum neurotoxin (BoNT) (BoNTA and BoNTE), which are proteins that are known to block the release of transmitters by targeting and entering nerve endings, where their proteases cleave and inactivate a protein, synaptosomal protein of Mr 25 000 (SNAP-25), that is essential for Ca2+-regulated exocytosis. Site-directed mutagenesis of Leu428 and Leu429 in BoNTA revealed that the remarkable longevity of its neuroparalytic action is attributable to a dileucine-containing motif. BoNTE acts transiently, because it lacks these residues, but is a superior inhibitor of transient receptor potential vanilloid type 1-mediated release of pain peptides from sensory nerves. The advantageous features of each serotype were harnessed by attaching the BoNTE protease moiety to an enzymically inactive mutant of BoNTA. The resultant purified composite protein could target motoneurons by binding to the BoNTA ectoacceptor and persistently produce BoNTE-truncated SNAP-25. As this enzyme lasted for more than 1 month (as compared with 5 days for BoNTE alone), such a dramatic extension in the lifetime of this BoNTE protease is attributable to a stabilizing influence of the BoNTA mutant. Most importantly, injecting this novel biotherapeutic into the foot pads of rats resulted in extended amelioration of inflammatory pain. Thus, a new generation of biotherapeutics has been created with the potential to give long-term relief of pain.