Botulinum toxins (BoNT) are zinc proteases (serotypes A-G) which cause flaccid paralysis through the cleavage of SNARE proteins within motor neurons. BoNT/A was originally organized into two subtypes, BoNT/A1 and BoNT/A2, which are approximately 95% homologous and possess similar catalytic activities. Subsequently, two additional subtypes were identified, BoNT/A3 (Loch Maree) and BoNT/A4 (657Ba), which are 81 and 88% homologous with BoNT/A1, respectively. Alignment studies predicted that BoNT/A3 and BoNT/A4 were sufficiently different from BoNT/A1 to affect SNAP25 binding and cleavage. Recombinant light chain (LC) of BoNT/A3 (LC/A3) and BoNT/A4 (LC/A4) were subjected to biochemical analysis. LC/A3 cleaved SNAP25 at 50% of the rate of LC/A1 but cleaved SNAPtide at a faster rate than LC/A1, while LC/A4 cleaved SNAP25 and SNAPtide at slower rates than LC/A1. LC/A3 and LC/A4 had similar K(m) values for SNAP25 relative to LC/A1, while the k(cat) for LC/A4 was 10-fold slower than that for LC/A1, suggesting a defect in substrate cleavage. Neither LC/A3 nor LC/A4 possessed autocatalytic activity, a property of LC/A1 and LC/A2. Thus, the four subtypes of BoNT/A bind SNAP25 with similar affinity but have different catalytic capacities for SNAP25 cleavage, SNAPtide cleavage, and autocatalysis. The catalytic properties identified among the subtypes of LC/A may influence strategies for the development of small molecule or peptide inhibitors as therapies against botulism.