The effects of mechanical stress on cultured muscle cells were examined with particular interest in myofibril assembly by using a cell-stretching system. We observed that formation and maintenance of cross-striated myofibrils in chick muscle cell cultures was suppressed in the media containing higher concentration of KCl, tetrodotoxin, or ML-9 (an inhibitor of myosin light chain kinase), but periodic stretching of myotubes for several days enabled formation of striated myofibrils just as in standard muscle cultures. However, ryanodine (a blocker of the Ca^{2 +} channel in sarcoplasmic reticulum) and BDM (an inhibitor of myosin-actin interaction) suppressed the stretch-induced myofibrillogenesis. We further found that stretching of myotubes causes quick and transient elevation of the intracellular Ca^{2 +} concentration and this elevation is disturbed by inhibition of Ca^{2 +} channels of sarcoplasmic reticulum and suppression of Ca^{2 +} influx from culture medium. These observations indicate that periodic stretching induces elevation of intracellular Ca^{2 +} concentration and that this elevation may be due to release of Ca^{2 +} from sarcoplasmic reticulum and Ca^{2 +} influx from outside of the cells. The increased Ca^{2 +} may activate actin-myosin interaction by interacting with troponin that is located along actin filaments and/or inducing phosphorylation of myosin light chains and thereby promote myofibril assembly.