Purpose: To examine force generation and duration of effect of botulinum toxin pretreatment, followed by injection of ricin-mAb35 in extraocular muscle. Methods: In normal adult rabbits, one superior rectus muscle was injected with either 5 units botulinum toxin or 1/50 maximally tolerated dose for rats (rMTD) of ricin-mAb35. Additional rabbits were first injected with 5 units botulinum toxin, and after 1, 2, or 4 weeks the same muscle was injected with either 1/10 or 1/50 rMTD ricin-mAb35. In each treatment group, the contralateral muscle was injected with an equal volume of saline. After 12 weeks (1/50 rMTD) or 6 months (1/10 rMTD), the rabbits were euthanized. Both SR muscles were removed and assayed physiologically, using an in vitro apparatus. Results: Twelve weeks after treatment with either botulinum toxin or immunotoxin alone, only ricin-mAb35-treated muscles were weaker than control muscles at tetanic stimulation frequencies. Pretreatment with botulinum toxin prior to injection with immunotoxin, especially at shorter intervals between injections, resulted in significant decreases in twitch and tetanic force generation compared with controls and muscles treated with ricin-mAb35 only or botulinum toxin only. At 6 months, force generation was decreased from control only in muscles treated with the higher dose of ricin-mAb35. Botulinum toxin pretreatment did not augment this effect at 6 months. Conclusions: Upregulation of postsynaptic nicotinic acetylcholine receptors caused by botulinum toxin pretreatment amplifies the reduction of force generation in extraocular muscle following secondary injection of the immunotoxin ricin-mAb35 within 3 months of treatment.