We developed a luciferase gene imaging system using a retrovirus for monitoring mesenchymal stem cell distribution. Retrovirus expressing firefly luciferase was infected into rat mesenchymal stem cells(rMSCs). rMSC-luc cells were excellent correlations between in vitro and in vivo bioluminescent activities and cell numbers. After systemic injections of cells in mice, luminescence activity was observed until six days, but was not detected in ischemic regions. After local injection with scaffold, luminescence activities were detected in injection sites over 3 weeks. This system could be a useful means for in vivo real time imaging of stem cell distributions, viability, and trafficking.