Stromal cell-derived factor-1alpha (SDF-1alpha) mediated mobilization and homing of stem cells showed promising potential in stem cell based tissue engineering and regenerative medicine. However local and sustained release of SDF-1alpha is indispensable for stem cell mediated regenerative process due to its short half-life under inflammatory conditions. In this study, a gene activated collagen substrate (GAC) was formed via assembly of plasmid encoding SDF-1alpha into a collagen substrate to create a microenvironment favoring stem cell homing. Local release of SDF-1alpha from the transfected cells on GAC and its effect on CD117(+) stem cell homing were investigated. Non-viral poly-ethyleneimine (25kDa PEI)/DNA complexes were mixed with rat tail collagen solution to form the GAC. Optimization of GAC was carried out based on collagen effects on the PEI/DNA complexes, viability and luciferase expression of COS7 cells on GAC. CD117(+) stem cells homing in response to SDF-1alpha local expression from transfected cells on GAC were investigated in a flow chamber in vitro and in a mouse hind limb model in vivo. The gene expression, migration of CD117(+) stem cells and the induced inflammation were investigated with immunostaining, reverse transcription polymerase chain reaction (RT-PCR) and H&E staining. The optimized parameters for GAC were DNA dosage 10 microg/cm(2), molar ratio of PEI nitrogen in primary amine to DNA phosphate (N/P ratio) 4 and mass ratio of collagen to DNA (C/D ratio) 1.0. It kept cell viability above 75% and transfection efficiency around 5.8 x 10(5) RLU/mg protein. GAC allowed the sustained gene release up to 60 days. GAC mediated SDF-1alpha gene release induced migration and homing of CD117(+) stem cells in vitro and in vivo significantly, and the inflammation of GAC reduced significantly two weeks after transplantation. GAC is a promising stem cell based therapeutic strategy for regenerative medicine.