The aim of this study was to present evidence for prolonged spontaneous firing in an anucleate axonal segment of an identifiable crayfish anal motoneuron L (AML) and to determine the initiation site of this firing. AML has its soma in the 6th abdominal ganglion (A6). By separating a nerve with the AML axon from A6 and the target muscle, various lengths of an anucleate AML axonal segment were procured. Then, AML activity was recorded extracellularly for 14–26 hr from the distal end of this axonal segment. This segment (n=19) exhibited spontaneous firing, which occurred without any stimulation 0.03–5.13 hr after the A6-cut and persisted tonically for 0.20–19.98 hr. During firing, the frequency augmented gradually, whereas the amplitude decreased gradually. There was no significant correlation between latency and duration of the firings. No correlation was noted between latency and length of the axonal segment or its size, or between duration and length or size. These results revealed that the anucleate AML axon itself can inherently generate prolonged firing. The delay in the appearance of AML impulses between the proximal and distal regions at the same axonal segment proved that the firing occurred proximally. There was no significant difference in delays between firing following the A6-cut and the spontaneous firing observed after the A6-cut. This suggests that the initiation site of the spontaneous firing is at the proximal end of the AML axonal segment, since the AML firing following the A6-cut occurs at its cut end.