Activities of the enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), which metabolize the neuromuscular transmitter acetylcholine, and malate and lactate dehydrogenase (MDH and LDH), enzymes of oxidative and glycolytic energy metabolism, respectively, were measured in the middle ear muscles of the cat. For comparison, the same enzyme activities were measured in extraocular muscle tissue and in three hindlimb muscles rich in either slow oxidative (soleus), fast glycolytic (white part of vastus lateralis), or fast oxidative glycolytic (plantaris) muscle fibers. ChAT and AChE activities were much higher in middle ear muscles than in hindlimb muscles, consistent with a denser neuromuscular innervation, as in extraocular muscles. By contrast, MDH and LDH activities were remarkably low in the middle ear muscles, lower than in any of the hindlimb muscles or the extraocular muscles. Denervation of the stapedius muscle by peripheral transection of the facial nerve resulted in decreases in all four enzyme activities without associated changes in the tensor tympani. Surgical ablation of the peripheral facial nerve supply to the stapedius muscle appears to be a feasible option for producing its denervation. The results suggest some rather specialized chemical characteristics for the middle ear muscles.