Kinetic studies for choline-O-acetyltransferase (CAT) (E.C. 126.96.36.199) and acetylcholinesterase (E.C. 188.8.131.52) were performed on serum, skin fibroblasts in culture, and biopsied sartorius muscle from normal and myasthenic subjects. There was a significant decrease of CAT activity per milligram of protein in myasthenic muscle compared to normal muscle, and there was no difference in acetylcholinesterase activity per milligram of protein in the same muscle homogenates. Substrate concentration curves for acetyl coenzyme A and CAT also showed a significant reduction in the maximum rate of product formation (Vmax) per milligram of protein between myasthenic and normal muscle. It is postulated that binding of substrate to CAT is being inhibited by an inhibitor present in muscle.