Introduction: Endothelial progenitor cells (EPCs) are not only involved in physiological neovascularization but also play an important role in wound healing, tissue regeneration and remodeling (e.g. in myocardial ischemia), and tumor angiogenesis. To exert some of these functions migration of the cells within the tissue is required. However, the kinetics of migration as well as the underlying mechanisms in vivo are still poorly understood. Therefore we aimed to develop a model to investigate migration of EPCs in vivo. Methods and Results: EPC migration in vivo was investigated in the dorsal skinfold chamber model in C57Bl/6 mice. Cultivated murine embryonic EPC (Hatzopoulos et al. 1998) were stimulated with SDF1-α (100ng/mL) to induce a migratory phenotype and labeled with Carboxyfluorescin (CFDA-SE 10µM) for 30 minutes before being detached from the culture plate using accutase. Immediately after preparation of the dorsal skinfold chamber model 10µL of the cell suspension (about 106 cells/mL) were injected into the dermal tissue in close apposition to a medium sized skin arteriole (diameter about 50µm) using a 30g needle. Application of the cells did not result in tissue edema or overt inflammation over an observation period of 3-4 days. According to expansion of the tissue volume covered by EPCs most cells remained intact and showed migratory activity as revealed by conventional intravital fluorescence microscopy as well as two-photon microscopy. While most cells migrated individually others formed larger aggregates within the tissue. Regular observations of migrating cells were possible by two-photon microscopy over several days. Conclusions: We established a model which allows us to characterize behavior and migration of EPCs in the perivascular tissue of the skin for several days. By using two-photon microscopy we were able to follow small changes and movements of single cells in the deeper layers of the tissue in a three-dimensional manner. Our model constitutes a potentially useful tool to investigate mechanisms involved in homing and migration of endothelial progenitor cells in response to different migratory stimuli.