The administration of naked DNA into animals is increasing as a research tool to develop DNA vaccine. To monitor the distribution and duration of gene expression of a DNA vaccine in living organisms, we used the naked DNA encoding firefly luciferase (Fluc) as an imaging reporter gene, and evaluated in vivo bioluminescent images in a murine model. We observed bioluminescence at the injection site and at inguinal lymph node from 10h to 24h post-injection when DNA vaccine encoding Fluc (pcDNA3.1-Fluc) was injected into the bilateral posterior flanks in mice. Fluc gene expressions at injection sites and unilateral posterior flank inguinal lymph node were also confirmed by RT-PCR. However, when pcDNA3.1-Fluc was injected into the mid-dorsum bioluminescent signals were observed at the injection site for up to 14 days post-injection, but no bioluminescent signals were detected in inguinal lymph nodes. Concurrent mRNA expressions of Fluc gene at injection sites but not at inguinal lymph nodes were confirmed by RT-PCR. These findings suggest that optical imaging using Fluc could be useful for monitoring the location, intensity and duration of gene expression of naked DNA vaccines in living animals non-invasively and repetitively.