This study was aimed to investigate the alteration of alpha-actin in three-dimensionally (3-D) cultured myocytes under cyclic tensile stress loading. Myocytes were collected from neonatal SD rat's lateral pterygoid muscle for primary cell culture. The third-passage cells were implanted and 3-D cultured in poly lactic-co-glycolic acid (PLGA) scaffold, and then subjected to cyclic tensile stress (0.5 Hz, 2,000 microstrain) for 0, 2, 4, 8, 12, and 24 h through a four-point bending strain system. The alpha-actin mRNA was investigated by semi-quantitative RT-PCR. The alpha-actin protein expression was examined by immunofluorescent cytochemistry, laser confocal scanning microscopy (LCSM), and image analysis technology. The dynamic adhesion of myocytes to PLGA scaffolds was investigated by fluorescence microscope and the viability of the myocytes was measured by MTT assay. After mechanical loading, the alpha-actin mRNA increased at 2 h and then declined. The alpha-actin protein expression kept increased until peaked at 12 h, but declined at 24 h. The time course changing of alpha-actin protein expression parallelled with that of cell adhesion ability. It is concluded that alpha-actin expression is probably associated with cell adhesion ability in myocytes subjected to mechanical stimulation.