Studies have been designed to examine the potential integration of DNA vaccines into the host cell genome. This is of concern because of the possibility of insertional mutagenesis resulting in the inactivation of tumor suppressor genes or the activation of oncogenes. The requirements for adequate testing were determined to be (1) a method to purify host cell genomic DNA from nonintegrated free plasmid, (2) a sensitive method to detect integrated plasmid in the purified genomic DNA, and (3) stringent methods to avoid contamination. These requirements were fulfilled by agarose-gel electrophoresis, the polymerase chain reaction, and separation of each activity with stringent handling procedures, respectively. An exploratory experiment was carried out in which mice were injected with 100 micrograms of vaccine plasmid DNA in each quadriceps. Examination of quadriceps and 12 other tissues at several time points failed to reveal any evidence of integration at a sensitivity level that could detect 1 to 7.5 integrations in 150,000 nuclei. A worst-case scenario determined that this would be at least 3 orders of magnitude below the spontaneous mutation frequency.