Brief treatment with 0.1 mM diisopropylfluorophosphate inhibited an average of 89% of the acetylcholinesterase (EC 3.1.1.7; acetylcholine hydrolase) activity of cultures of chick embryo muscle. As long as protein synthesis occurred, an average of 78% of the activity returned within 4 hr. Newly synthesized acetylcholinesterase did not stain cytochemically, was rapidly and extensively degraded or released in the presence of 10 muM cycloheximide, and consisted mainly of low-molecular-weight forms. Acetylcholinesterase activity first appeared around the nucleus, about 4 hr after treatment with diisopropylfluorophosphate, and then spread to the rest of the cell about the time release of acetylcholinesterase was detected in the medium. With time, more and more of the enzyme was retained in the cells after treatment with cycloheximide, and the proportions of low-molecular-weight forms decreased and high-molecular-weight forms increased. The results suggest that newly synthesized acetylcholinesterase undergoes an orderly process of binding, movement, and assembly in diisopropylfluorophosphate treated, and probably also in untreated, embryo muscle fibers.