Synthesis and degradation of acetylcholine receptors in rat skeletal muscles were measured in organ culture. The rate of de novo biosynthesis and incorporation of acetylcholine receptors into extrajunctional membranes of denervated muscles was measured by determining the rate of appearance of [1] [2H, 13C, 15N]-acetylcholine receptors when muscles were cultured in medium containing [1] [2H, 13C, 15N]-amino acids. Denervated extensor digitorum longus and soleus muscles were found to synthesize new receptors for several days in organ culture at an average rate of 1.4%/h. The degradation rates for extrajunctional and junctional acetylcholine receptors were estimated by irreversibly labeling acétylcholine receptors on muscles with radioactive iodinated α-bungarotoxin and measuring the rate of release into the culture medium of mono- and di-iodotyrosine, breakdown products of the radioactive α-bungarotoxin. The rates of this proteolytic process yielded average lifetimes of 22 h and 13 days for [125I]α-bungarotoxin bound to extrajunctional and junctional receptors, respectively, probably reflecting the average lifetimes of the acetylcholine receptors. Electrical stimulation at 100Hz for 1 s every 80s, producing visible contraction, but not maximal tetanic tension, barely altered the rate of incorporation of new acetylcholine receptors into the extrajunctional plasma membrane of extensor digitorum longus and soleus muscles, even when the stimulation continued for 5 days. Supra-maximal stimulation, resulting in maximal tetanic tension, with the same stimulation pattern produced a rapid decline of 10–20% in the rate of new receptor production and a corresponding decline in overall protein synthesis. Stimulation beyond 18–24 h (up to 68 h) resulted in a further decrease in new receptor production to about 30% of the control rate, but not more. Stimulation for longer than 16 h produced less than a 5–10% decrease in overall protein synthesis, compared with control muscles. The same pattern of electrical stimulation, producing maximal tetanic tension, had no effect on the apparent degradation rate of extrajunctional receptors in denervated muscles. Our results show that denervated adult muscle can be maintained in organ culture for at least 1 week, and that the muscles in culture will continue to degrade acetylcholine receptors and to synthesize new receptors, even when electrically stimulated for 5 days. The significance of this study is that electrical stimulation, producing frequent tetanic contractions, can affect extrajunctional acetylcholine receptor metabolism by selectively decreasing de novo synthesis, the receptor degradation remaining unchanged. The precise control point in the biosynthetic process has yet to be determined. However, our results indicate that electrically induced activity does exert a regulating influence, relatively rapidly, on extrajunctional acetylcholine receptor metabolism on muscles maintained in vitro.