The type A neurotoxin produced by Clostridium botulinum is a potent neuromuscular blocking agent which causes paralysis by preventing the release of neurotransmitter from motor neurons. This property has resulted in the use of the toxin in the treatment of a number of neuromuscular diseases involving muscle spasms. At present, the only recognised assay to estimate accurately the potency of botulinum toxin in clinical preparations is bioassay, in which lethality is used as the endpoint. Such bioassay is inherently variable and large interlaboratory variability has been reported, highlighting problems for standardisation of activity in the absence of any commonly used reference preparation. In the present study, we have confirmed that many different assay conditions can affect potency estimates of clinical formulations of type A botulinum toxin. Further, our studies indicate that different preparations, because of their unique formulation and stability, are differentially affected by some of these assay conditions and that these differences might well contribute to the differences observed in their clinical use.