We determined levels of mRNA encoding acetylcholinesterase (AChE) in muscles of rats subjected to chronic enhancement of neuromuscular activation. After 8 wk of voluntary wheel running, extensor digitorum longus (EDL) muscles displayed a 72% increase in total AChE activity as a result of a selective threefold increase in the G4 content. Soleus muscles, on the other hand, exhibited a 30% decrease in A12 while displaying a small (33%) increase in total AChE activity. These enzymatic adaptations were paralleled by increases in the levels of AChE mRNAs in both EDL (32%; P < 0.03) and soleus (42%; P < 0.02) muscles. In addition, compensatory hypertrophy of the plantaris muscle increased total AChE activity by 75%. This change was reflected by an elevation in all AChE molecular forms with A12 (89%) and A8 (179%) showing the most prominent increases. Similar to exercise-trained muscles, hypertrophied plantaris muscles displayed an increase in AChE transcripts (25%; P < 0.04). These results indicate that increases in neuromuscular activity modulate expression of the AChE gene in vivo and suggest the involvement of pretranslational regulatory mechanisms in the adaptive response of AChE to enhanced neuromuscular activation.