The classical model of 17β-estradiol action has been traditionally described to be mediated by the estrogen receptor (ER) localized exclusively in the nucleus. However, there is increasing functional evidence for extra nuclear localization of ER. We present biochemical, immunological and molecular data supporting mitochondrial-microsomal localization of ERα in the C2C12 skeletal muscle cell line. We first established [3H]17βestradiol binding characteristics in whole cells in culture. Specific and saturable [3H]17βestradiol binding sites of high affinity were then detected in mitochondrial fractions (Kd = 0.43 nM; Bmax = 572 fmol/mg protein). Immunocytological studies revealed that estrogen receptors mainly localize at the mitochondrial and perinuclear level. These results were also confirmed using fluorescent 17βestradiol-BSA conjugates. The immunoreactivity did not translocate into the nucleus by 17β-estradiol treatment. Western and Ligand blot approaches corroborated the non-classical localization. Expression and subcellular distribution of ERα proteins were confirmed in C2C12 cells transfected with ERα siRNA and by RT-PCR employing specific primers. The non-classical distribution of native pools of ERα in skeletal muscle cells suggests an alternative mode of ER localization/function. J. Cell. Biochem. 104: 1254–1273, 2008. © 2008 Wiley-Liss, Inc.