The turnover of acetylcholine receptors (AChRs) was studied at developing motor endplates in embryonic rat diaphragm and at newly formed ectopic endplates in soleus muscles of adult rats. After the receptors were labeled in situ with 125I-alpha-bungarotoxin, the rate of loss of bound toxin was determined by autoradiography of single muscle fibers and was used to calculate the turnover time of AChRs. A new, convenient method for preparing large numbers of single muscle fibers is described. AChRs in extrajunctional regions of embryonic diaphragms turn over with a short half-time (24 hr) similar to that of AChRs in cultured myotubes and of extrajunctional AChRs in denervated adult muscle. AChRs in newly formed clusters in developing muscle and in ectopically innervated adult muscle also turn over with short half-times. Within a few days, however, the turnover time increases to values characteristic of adult junctional receptors (6-10 days). Transection of the nerve at newly formed ectopic endplates prevents the change. The metabolic stabilization of AChRs at motor endplates in rat muscles is thus a relatively early event in synapse formation; it coincides neither with the clustering of receptors, which precedes it, nor with the decrease in AChR channel open time which has been shown to occur postnatally.