DNA degradation is a fundamental problem for any gene therapy or genetic immunization approach, since destruction of incoming genes translates into loss of gene expression. To characterize the biology of DNA degradation after naked DNA injection, the location and levels of tissue nucleases were assessed. Extracts from the serum, kidney, and liver of mice had high levels of calcium-dependent endonuclease activity. High levels of acidic endonuclease activity were identified in the spleen, liver, kidney, and skin with little activity in skeletal or cardiac muscle. Relatively little exonuclease activity was observed in any tissue. The presence of endonucleases in the skin and muscle mediated degradation of 99% of naked DNA within 90 min of injection. This degradation most likely occurred in the extracellular space upstream of other cellular events. Despite this massive destruction, gross tissue nuclease levels did not determine skin-to-muscle transfection efficiency, or site-to-site transfection efficiency in the skin. While gross tissue nuclease levels do not appear to determine differences in transfection efficiency, the presence of robust tissue nuclease activity still necessitates that massive amounts of DNA be used to overcome the loss of 99% of expressible DNA. In addition to destroying genes, the nucleases may play a second role in genetic immunization by converting large plasmids into small oligonucleotides that can be taken up more easily by immune cells to stimulate CpG-dependent Th1 immune responses. For genetic immunization, vaccine outcome may depend on striking the right balance of nuclease effects to allow survival of sufficient DNA to express the antigen, while concomitantly generating sufficient amounts of immunostimulatory DNA fragments to drive Th1 booster effects. For gene therapy, all nuclease effects would appear to be negative, since these enzymes destroy gene expression while also stimulating cellular immune responses against transgene-modified host cells.