AIM: MyoD, myogenin, proliferating cell nuclear antigen (PCNA) and cyclin-dependent kinase inhibitor p21 (p21) proteins are key molecules in inducing the growth of myogenic cells in vitro. However, it has not been determined which cell types express these factors in hypertrophying skeletal muscles in vivo. METHODS: Using immunohistochemical techniques, we examined the spatial and temporal expression patterns of MyoD, myogenin, PCNA and p21 proteins in functionally overloaded rat plantaris muscles induced by ablation of the soleus and gastrocnemius muscles. RESULTS: MyoD and myogenin were detected in myonuclei located inside the dystrophin-positive plasma membrane of myofibres, m-cadherin-positive satellite cell nuclei and nuclei located in the interstitial spaces between myofibres on days 1, 3, 5 and 7 post-surgery. Entry of satellite cells into the cell cycle was indicated by the expression of PCNA on day 3 post-surgery, and withdrawal from the cell cycle was observed by the expression of p21 in satellite cell nuclei on day 5 post-surgery. However, the expression of both PCNA and p21 in satellite cell nuclei disappeared on day 7 post-surgery. CONCLUSION: These results indicate that proliferated satellite cell-derived myoblasts and undefined myogenic cells located in the interstitial spaces may contribute to an increase in myonuclear number and/or hyperplasia. Furthermore, we provide evidence that all of myonuclei, satellite cells and undefined myogenic cells express both MyoD and myogenin proteins. These results suggest that continual expression of MyoD and myogenin proteins in these cells is an essential molecular event which induces the successful hypertrophy of skeletal muscles.