Changes in neuromuscular activity are known to modify neuromuscular transmission. In this report, we describe changes in the activities of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) in response to decreased and increased activity of skeletal muscles in the rat. Disuse atrophy of gastrocnemius muscles was produced by surgical pinning of the hind limb. Overuse hypertrophy of the plantaris muscle was produced by severing the tendon of the gastrocnemius and soleus muscles. At various times after production of disuse or overuse, the activities of CAT and AChE were measured by radiometric assay. In disused muscles, CAT activity per muscle decreased markedly (50% of control by day 7), while AChE activity per muscle decreased to a smaller extent (80% of control by day 7). The K m of CAT with respect to acetyl CoA did not change in homogenates of atrophic muscles (1 × 10 −4 m), suggesting that the loss of activity was not due to alteration of enzyme protein, but rather to decreased amounts of enzyme. Overuse of muscle resulted in an increase in the activities of CAT (120% of control by day 8) and AChE (142% of control by day 8). Thus, use and disuse may influence neuromuscular transmission by regulating the production of enzymes involved in the synthesis and degradation of acetylcholine.