In addition to skeletal muscle paralysis, type A botulinum toxin commonly causes sprouting of motor axons in various experimental whole-animal systems. The use of type A botulinum toxin in clinical treatment of muscle spasm disorders is becoming increasingly popular. The eventual, unwanted return of involuntary activity in the treated muscles may be a consequence of such axon sprouting. We have developed a coculture model allowing the quantification of botulinum toxin-induced sprouting that shows promise for future studies on its mechanism and control. Chick embryo ciliary ganglion motor neurons were cocultured with chick leg muscle cells. The presence of type A botulinum toxin in the coculture medium was correlated with significantly increased branching frequency of neurites. Toxin-increased branching frequency occurred even when the neurons and muscle cells were separated from each other on the culture dishes, suggesting a presynaptic effect of toxin. Cocultures incubated in the presence of curare, a post-synaptic blocker, had control levels of neurite branching, ruling out the possibility that simple synaptic blockade causes sprouting but again supporting the hypothesis of a pre-synaptic activity of botulinum toxin.