In vivo gene therapy was attempted using poly-(D,L-lactic-co-glycolic acid) microspheres containing the interleukin-12 gene (p2CMVmIL 12) in colon adenocarcinoma (CT-26)-bearing Balb/c mice. Treatment with IL-12-loaded microspheres inhibited tumor growth significantly compared to a control group treated with free IL-12 DNA, though the degree of tumor inhibition was not dependent on the amount of IL-12 gene loaded. Combining gene therapy with 5-fluorouracil (50 mg/kg) treatment further inhibited tumor growth compared with gene therapy alone. The results indicate that microsphere formulations of pDNA may provide an efficient gene delivery system.