We sought to determine whether in vivo electroporation might overcome the requirement for strong transcriptional promoters in plasmid vaccines. We tested plasmid vaccines containing viral promoters that exhibited a broad range of transcriptional activities by in vitro assay. In contrast to the in vitro observations, no difference in gene expression was measured in vivo 24h following injection when the plasmids were introduced via electroporation. Despite the similarities in gene expression in vivo, the cellular and humoral responses elicited by these plasmid vaccines were dramatically different and correlated more closely with the promoter activity measured in vitro. When antigen production in vivo was measured over a longer period following injection, significant differences in gene expression became apparent after 2-3 days and the differences in gene expression at day 7 correlated well with plasmid immunogenicity. These studies reconfirm the importance of antigen production for effective plasmid vaccination and demonstrate that the duration of gene expression should also be considered when designing plasmid vaccines.