Excitation-contraction coupling is the physiological mechanism whereby an electrical signal detected by the dihydropyridine receptor, is converted into an increase in [Ca2+], via activation of ryanodine receptors. Mutations in RYR1, the gene encoding ryanodine receptor 1, are the underlying cause of various congenital myopathies including Central Core Disease, Multiminicore disease, some forms of Centronucleal myopathy and Congenital Fiber Type disproportion. Interestingly, patients with recessive but not dominant RYR1 mutations show a significant reduction of ryanodine receptor protein in muscle biopsies as well as ophthalmoplegia. In order to understand why the patients with recessive RYR1 mutations specifically showed involvement of the extraocular muscles, we investigated the excitation-contraction coupling machinery in biopsies obtained from “normal” patients undergoing resection eye surgery. Our results show that the major proteins involved in skeletal muscle excitation-contraction coupling are expressed differently in human extraocular muscles compared to leg muscles. In particular the transcripts encoding ryanodine receptor 3, cardiac calsequestrin and the alfa 1 subunit of the cardiac dihydropyridine receptor were upregulated by at least 100 fold, whereas ryanodine receptor 1 and the alfa 1 subunit of the skeletal dihydropyridine receptor were reduced 10 fold. Myotubes obtained from extraocular muscle biopsies also exhibited changes in their calcium homeostasis, and particularly the resting [Ca2+] was lower and the depolarization induced Ca2+ influx was 3 fold higher compared to that observed in leg muscle-derived myotubes. These results indicate that extraocular muscles have a different mode of calcium handling; moreover, the presence of ophthalmoplegia in patients with recessive RYR1 mutations is most likely due to the lower endogenous levels of RyR1 expressed by the extraocular muscles.