Slow antigravity muscles differ from fast muscles with regard to load bearing performed during contraction. We examined the importance of load bearing in regulation of acetylcholinesterase (AChE) expression in slow and fast rat muscles. The levels of AChE mRNA in the slow soleus muscles are about 30% of those in the fast extensor digitorum longus (EDL) muscles. In the soleus muscles unloaded for 8 days by hindlimb suspension, AChE mRNA levels were not significantly different from those in the control soleus muscles. In the suspended animals, AChE transcripts in the EDL muscles decreased to about 80% of control levels. Reduction of the resting muscle tension by joint fixation did not significantly affect the levels of AChE mRNA in the unloaded soleus muscles. Phasic high-frequency electrical stimulation of the unloaded soleus muscles via the sciatic nerve increased their AChE mRNA levels to about 50% of those in the EDL muscles. The levels observed after phasic stimulation were significantly higher than those after low-frequency tonic stimulation, indicating the importance of muscle activation pattern for AChE regulation also in the absence of load bearing. The AChE mRNA levels in the soleus muscles overloaded for 8 days by synergist muscle ablation increased significantly to about 50% of those in the EDL muscle. The load bearing during muscle contraction seems to be a relatively unimportant extrinsic factor in the regulation of the AChE mRNA levels in muscle fibers, except when an increased load induces muscle hypertrophy accompanied by the fusion of satellite cells with the muscle fibers. © 2002 Wiley-Liss, Inc.