Botulinum neurotoxins (BoNT), produced by the anaerobic bacterium Clostridium botulinum, cause severe neuroparalytic disease and are considered the most toxic biological agents known. While botulism is rare in the U.S. it often is fatal if not treated quickly, and recovery is long, requiring intensive treatment. BoNT is synthesized as a 150 kDa precursor protein (holotoxin), which is then enzymatically cleaved to form two subunit chains linked by a single disulfide bond. The 'gold standard' for BoNT detection relies on a mouse bioassay. This is a time consuming (up to 4 days) assay and it lacks specificity, however, it gives a sensitivity (mouse LD(50)) of approximately 10 pg mL(-1). Most BoNT immunoassays are much less sensitive. In this study we describe the development of four high-affinity (dissociation constants (Kd's) in the low pM range) monoclonal antibodies (mAbs) that specifically bind BoNT serotype A (BoNT/A). These antibodies, designated F1-2, F1-5, F1-40, and F2-43 are IgG1 subclass mAbs with kappa light chains and they specifically bind BoNT serotype A. Western blot analyses following SDS-PAGE demonstrate that mAbs F1-2 and F1-5 bind the 100 kDa heavy chain subunit and that mAb F1-40 binds the 50 kDa light chain. The fourth antibody demonstrated strong binding to the 150 kDa holotoxin in the ELISA and on Western blots following electrophoresis on native gels. However binding in Western blot studies was not observed for mAb F2-43 following SDS-PAGE. A highly sensitive sandwich ELISA, capable of detecting as little as 2 pg/mL BoNT/A was developed using mAbs F1-2 and F1-40. Such an assay represents a realistic, high sensitivity alternative to the mouse bioassay.