Prolonging gene expression in skin using safe, nonviral gene delivery techniques could impact skin regeneration and wound healing, decrease infection, and potentially improve the success of tissue-engineered skin. To this end, an injectable, agarose-based delivery system was tested and shown to prolong nonviral gene expression in the skin. DNA was compacted with polylysine to improve DNA stability in the presence of nucleases. Up to 25 microg of compacted luciferase plasmid with or without agarose hydrogel was injected intradermally in rodents. Bioluminescence imaging was used for longitudinal, noninvasive monitoring of gene expression in vivo for 35 days. Injections of DNA in solution produced gene expression for only 5-7 days, whereas the sustained release of compacted DNA from the agarose system prolonged expression, with more than 500 pg (20% of day 1 levels) of luciferase per site for at least 35 days. Southern blotting confirmed that the agarose system extended DNA retention, with significant plasmid present through day 7, as compared with DNA in solution, which had detectable DNA only on day 1. Histology revealed that agarose invoked a wound-healing response through day 14. Tissue-engineering and wound-healing applications may benefit from the agarose gene delivery system.