Given that adult adipose tissue is an abundant, accessible and safe source of stem cells, the use of adipose-derived stem cells (ADSCs) provides a promising approach in ischemic stroke. The delivery route, however, for transplantation of ADSCs in clinical application remains controversial regarding the time window, cell type, safety issues, 'first pass' effect and therapeutic effect. To determine the optimal administration route in transplantation of ADSCs, we compared the therapeutic effect of the three mainly used administration routes of ADSCs in a middle cerebral artery occlusion (MCAO) rat model. Cells isolated from the adipose tissue of adult rodents were differentiated and characterized in vitro, and further transplanted in vivo by intravenous, intra-arterial or intra-ventricular delivery. The infarct volume, expression of neurotrophic factors and the neurobehavioral improvements were evaluated after the equal dose of BrdU labeled ADSCs transplantation. Our results indicated that the equal dose of ADSCs delivered intravenously were effective in improving the neurological outcome and reducing the infarct volume after ischemic brain injury in long term duration in contrast to intra-arterial and intra-ventricular delivery. At 1-7 days after transplantation, the increased expression levels of BDNF, VEGF, bFGF, Bcl-2, IL-10 and decreased levels of caspase-3 and TNF-alpha in the intra-ventricular and intra-arterial groups were significant in contrast to the intravenous group. There was no significant difference among the three groups after 7 days. Our findings suggest that compared with the intra-ventricular delivery, intravascular injection allows higher dose injection with fewer invasions and appears to be optimal in application with regard to therapeutic efficacy, safety and feasibility. (C) 2014 Elsevier GmbH. All rights reserved.