OBJECTIVE: Proprioception from extraocular muscles (EOMs) is important for proper visually-guided behavior. Surprisingly, the classical proprioceptor pair (muscle spindles and Golgi tendon organs) is absent in the EOMs of most mammals and instead, palisade endings have been found in virtually all species analyzed so far. Due to their widespread distribution it is believed that palisade endings are candidates for EOM proprioception. We have analyzed the molecular profile and the central connection of palisade endings. METHODS: Whole mount of cat EOMs were triple labeled with neuronal markers, including markers for cholinergic axons, exocytosis proteins, and acetylcholine receptors. In anterograde tracing experiments, neuronal tracer was injected into the EOM motor nuclei. RESULTS: Palisade endings were formed by nerve fibers that, coming from the muscle, extended into the tendon where they made a u-shaped turn to approach the muscle tendon junction. Recurrent nerves divided into axonal branches and terminal varicosities to form palisade endings at the tip of single muscle fibers. Molecular analyses showed that palisade endings had a cholinergic phenotype because they expressed choline acetyl transferase, the synthesizing enzyme of acetylcholine. Additionally, palisade ending expressed SNAP25, synaptobrevin, synaptotagmin, complexin, and syntaxin, all of which are key proteins involved in neurotransmitter release. Acetylcholine esterase, the degrading enzyme of acetylcholine was absent in palisade ending and no receptors for cholinergic transmission were associated with palisade endings as demonstrated by the absence of alpha-bungarotoxin signals. Following neuronal tracer injection in the EOM motor nuclei, palisade endings were labelled. CONCLUSION: Our findings show that palisade endings are cholinergic, have an exocytosis machinery for neurotransmitter (acetylcholine) release and originate from the EOM motor nuclei. However, no receptors for cholinergic transmission are associated with palisade endings suggesting that palisade ending belong to an effector system that is different from that in other skeletal muscles.