DNA vaccination has proven to be effective against a number of tumours and microbial diseases. As DNA vaccines are unable to replicate, plasmid copy number per cell is dependent on in vivo transfection efficiency, which is usually quite low. Consequently, immune responses generated are likely to be sub-optimal due to low antigen expression levels in transfected cells. During this study, replicating DNA vaccines delivered intra-epidermally by gene gun, were assessed for their ability to more efficiently generate immune responses in mice. The data demonstrate that, using a polyoma virus-based system of replication, 10-fold less DNA expressing the haemagglutinin gene of influenza virus, was required to stimulate a humoral immune response, compared to an equivalent non-replicating vaccine. This observation suggests that the use of replicating DNA vaccines in some delivery systems may enhance the effectiveness of immune responses.