The aim of the present study was to investigate the activation of estrogen response elements (EREs) by estrogen and muscle contractions in rat myotubes in culture and to assess whether the activation is dependent on the estrogen receptors (ERs). In addition, the effect of estrogen and contraction on the mRNA levels of ER and ER was studied to determine the functional consequence of the transactivation. Myoblasts were isolated from rat skeletal muscle and transfected with a vector consisting of sequences of EREs coupled to the gene for luciferase. The transfected myoblasts were then differentiated into myotubes and subjected to either estrogen or electrical stimulation. Activation of the ERE-sequence was determined by measurement of luciferase activity. The results show that both ER and ER are expressed in myotubes from rat. Both estrogen stimulation and muscle contraction increased (p < 0.05) transactivation of the ERE-sequence and enhanced ER mRNA, whereas ER was unaffected by estrogen and attenuated (p < 0.05) by muscle contraction. Use of ER antagonists showed that whereas the estrogen-induced transactivation is mediated via ERs, the effect of muscle contraction is ER-independent. The muscle contraction induced transactivation of ERE and increase in ER mRNA was instead found to be MAP kinase (MAPK) dependent. This study demonstrates for the first time that muscle contractions have a similar functional effect as estrogen in skeletal muscle myotubes, causing ERE activation and an enhancement in ER mRNA. However, in contrast to estrogen, the effect is independent of ERs and dependent on MAPK, suggesting activation via the estrogen related receptor (ERR).