Cell therapies from various sources have been under intense research in stroke. Efficient homing of the cells to the injured brain without complications is necessary to realize the therapeutic potential of cell therapy. Intra-arterial (IA) infusion of cells bypasses the filtering organs and directs the cells to the target area more efficiently. Here we studied the biodistribution of human bone marrow-derived mesenchymal stromal/stem cells (BMMSCs) after a direct infusion into the external carotid artery (ECA) in rats. Cells, which were cultured without animal-derived agents and also treated with a proteolytic enzyme to transiently modify cell surface adhesion proteins, were infused 24h after transient middle cerebral artery occlusion (MCAO). SPECT imaging was used immediately after cell infusion and 24h thereafter to track (111)In-oxine-labeled BMMSC in sham-operated and MCAO rats. IA infusion of BMMSCs in rats resulted in immediate cell entrapment in the brain, but the majority of the signal disappeared during the next 24h and relocated to the internal organs. In MCAO rats, radioactivity counts 24h after infusion were higher in the ischemic hemisphere compared to the contralateral hemisphere. Our results showed that IA infusion through ECA is a safe and efficient administration route for BMMSCs resulting in a transient localization of cells in the rat brain.