At the mouse neuromuscular junction (NMJ), there are two distinct cholinesterases (ChE): acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Until now, it has been difficult to determine the precise localization of BChE at the NMJ. In this study, we use a modification of Koelle's method to stain AChE and BChE activity. This method does not interfere with fluorescent co-staining, which allows precise co-localization of ChE and other synaptic molecules at the NMJ. We demonstrate that AChE and BChE exhibit different localization patterns at the mouse NMJ. AChE activity is present both in the primary cleft and in the secondary folds, whereas BChE activity appears to be almost absent in the primary cleft and to be concentrated in subsynaptic folds. The same localization for BChE is observed in the AChE-knockout (KO) mouse NMJ. Collagenase treatment removed AChE from the primary cleft, but not from secondary folds in the wild-type mouse, whereas in the AChE-KO mouse, BChE remains in the secondary folds. After peripheral nerve injury and regeneration, BChE localization is not modified in either normal or KO mice. In conclusion, specific localization of BChE in the secondary folds of the NMJ suggests that this enzyme is not a strict surrogate of AChE and that the two enzymes have two different roles.