Cultures of 11 day old chick embryo pectoral muscle were used to study the effects of direct electrical stimulation and neurochemicals such as acetylcholine, acetyl-beta-methylcholine, tetrodotoxin, and d-tubocurarine on the acetylcholinesterase levels of muscle. The results suggest that excitation-contraction is an important factor in regulation of muscle acetylcholinesterase. Tetrodotoxin, acetylcholine and its analog acetyl-beta-methylcholine increased acetylcholinesterase levels and reduced spontaneous contractions. D-tubocurarine blocked the increase in acetylcholinesterase and the decrease in spontaneous contractions caused by acetyl-beta-methylcholine. Electrical stimulation decreased acetylcholinesterase and increased muscle contractions in normal and in diisopropylfluorophosphate treated cultures. Tetrodotoxin also affected the morphology of the muscle cells, as if it adversely affected normal growth and differentiation. Electrical stimulation did not increase muscle creatine kinase.