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In order to determine the role of acetylcholine (ACh) transmission in neurotrophic regulation of dynamic properties of muscle, the effects of botulinum toxin treatment were compared with those of denervation. The extensor digitorum longus (EDL) and soleus muscles of rats were either denervated or injected with botulinum toxin. At times up to 25 days the isometric properties of these muscles were determined. 2. Both botulinum treatment and denervation produced progressive slowing of the time to peak of the twitch (TPT) and half-relaxation time of the twitch (1/2 RT), which was more pronounced in the EDL than in the soleus. 3. Both treatments produced slowing of the relaxation curve following tetanic contraction, more marked in the EDL than in the soleus muscle. This indicates a slowing of relaxation, and suggests a prolongation of the active state of the muscle. 4. The maximum rate of rise of the tetanus did not change significantly in the EDL and soleus muscles after botulinum treatment or denervation. This suggests that there is no major change in the speed of contraction under conditions of botulinum treatment or denervation. 5. The changes produced by botulinum treatment and denervation were virtually identical in all parameters tested. This is interpreted meaning that cholinergic transmission (including muscle usage), or some other factor closely related to cholinergic transmission, accounts for the motor nerve's trophic influence in maintaining these dynamic properties of skeletal muscles.