Excitation contraction coupling is the physiological mechanism occurring in muscle cells whereby an electrical signal sensed by the dihydropyridine receptor located on the transverse tubules is transformed into a chemical gradient (Ca2+ increase) by activation of the ryanodine receptor located on the sarcoplasmic reticulum membrane. In the present investigation we characterized for the first time the excitation contraction coupling machinery of an immortalized human skeletal muscle cell line. Intracellular Ca2+ measurements showed a normal response to pharmacological activation of the ryanodine receptor whereas super resolution structured illumination microscopy (3D-SIM) revealed a low level of structural organization of ryanodine receptors and dihydropyridine receptors. Interestingly, the expression levels of several transcripts of proteins involved in calcium homeostasis and differentiation indicate that the cell line has a phenotype closer to that of slow twitch than fast twitch muscles. These results point to the potential application of such human muscle-derived cell lines to the study of neuromuscular disorders; in addition they may serve as a platform for the development of therapeutic strategies aimed at correcting defects in calcium homeostasis due to mutations in genes involved in calcium regulation.