Bacterial neurotoxins are now being used routinely for the treatment of neuromuscular conditions. Alternative assays to replace or to complement in vivo bioassay methods for assessment of the safety and potency of these botulinum neurotoxin-based therapeutic products are urgently needed. Advances made in understanding the mode of action of clostridial neurotoxins have provided the basis for the development of alternative mechanism-based assay methods. Thus, the identification of SNAP-25 (synaptosomal-associated protein of molecular mass 25 kDa) as the intracellular protein target which is selectively cleaved during poisoning by botulinum neurotoxin type A (BoNT/A) has enabled the development of a functional in vitro assay for this toxin. Using recombinant DNA methods, a segment of SNAP-25 (aa residues 134-206) spanning the toxin cleavage site was prepared as a fusion protein to the maltose-binding protein in Escherichia coli. The fusion protein was purified by affinity chromatography and the fragment isolated after cleavage with Factor Xa. Targeted antibodies specific for the N and C termini of SNAP-25, as well as the toxin cleavage site, were prepared and used in an immunoassay to demonstrate BoNT/A endopeptidase activity towards recombinant SNAP-25 substrates. The reaction required low concentrations of reducing agents which were inhibitory at higher concentrations as were metal chelators and some inhibitors of metallopeptidases. The endopeptidase assay has proved to be more sensitive than the mouse bioassay for detection of toxin in therapeutic preparations. A good correlation with results obtained in the in vivo bioassay (r = 0.95, n = 23) was demonstrated. The endopeptidase assay described here may provide a suitable replacement assay for the estimation of the potency of type A toxin in therapeutic preparations.