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The acetylcholinesterase (AChE) in rat diaphragms was labelled by intravenous injection of echothiophate in order to evaluate the turnover of AChE in innervated and denervated muscle in vivo. Echothiophate diethylphosphorylates AChE thereby inactivating it. Labelled (diethylphosphorylated) enzyme is rapidly and quantitatively reactivated with 1-methyl-2-hydroxyiminomethylpyridinium (2-PAM), so labelled (diethylphosphorylated) AChE was conveniently measured as 2-PAM-reactivatable AChE activity. In homogenates in vitro, label is lost spontaneously (diethylphosphorylated AChE spontaneously reactivates) with a half-time of 27 h. In innervated diaphragm, labelled non-end-plate AChE is lost with a half-time of 13 h. When correction is made for the spontaneous loss of label on the basis of in vitro measurements, this data indicates that non-end-plate AChE turns over with a half-time of about 26 h. In innervated diaphragm, labelled end-plate-specific AChE is lost more slowly than non-end-plate AChE and at a rate essentially identical to the rate of spontaneous loss of label in vitro. The rate of loss of labelled non-end-plate AChE is essentially identical in 18 h denervated and in paired innervated diaphragms. The rate of loss of labelled end-plate-specific AChE is significantly faster in 18 h denervated diaphragms than in paired innervated diaphragms. On the basis of these observations, hypotheses concerning the mechanisms of the denervation-induced decreases in non-end-plate and end-plate-specific AChE are formulated and discussed.