Fiber diameter variability, acetylcholinesterase staining properties, and average fiber diameter were determined 5 weeks after varying doses of botulinum A toxin were administered into albino rabbit longissimus dorsi muscle. The average fiber diameter within the muscle appeared to be a function of the dose of botulinum toxin injected. Fiber diameter variability correlated with the dose of botulinum toxin administered. Both fiber diameter variability and acetylcholinesterase spread characteristics showed a distinct diffusion gradient over a defined field within a muscle. At lower doses (1 IU), collapse of the diffusion gradient occurred over a 15-30-mm segment of muscle. At higher doses (5-10 IU), diffusion of botulinum A toxin effect occurred throughout the entire muscle with no apparent end point. This study demonstrated that botulinum A toxin produces a gradient of denervation in a given muscle and that both the magnitude of denervation and the extent of the gradient are dose dependent. Furthermore, both muscle fiber diameter variability and acetylcholinesterase staining were useful as measures of chemodenervation.