Acetylcholinesterase inhibition with neostigmine in the isolated rat phrenic nerve-hemidiaphragm preparation induced axonal backfiring and repetitive compound muscle action potentials following single nerve stimulation. The duration of backfiring and the repetitive compound muscle action potentials did not exceed 55 ms. With repetitive nerve stimulation at frequencies ranging from 1 to 50 Hz, backfiring was present only with the first stimulus and the amplitude of the second compound muscle action potential was maximally reduced, while the subsequent responses recovered gradually. However, the amplitudes of the concomitant antidromic nerve action potentials remained unchanged during the entire train of stimulation. Lack of nerve action potential amplitude changes and the short duration of backfiring of the first nerve action potential exclude a collision phenomenon of backfiring with the nerve action potential induced by the second stimulus. Moreover, the duration of the repetitive compound muscle action potentials did not exceed the duration of backfiring. Therefore, the prolongation of the muscle membrane refractory period by reexcitation following backfiring cannot explain the decrement of the second compound muscle action potential.