Rapid, large-volume injection, or so-called hydrodynamic injection, of naked plasmid DNA gives high transgene expression in mouse liver, and this method has been applied to liver-directed gene transfer in humans with slight modifications. To prove that injection-induced biological changes are involved in hydrodynamic injection-induced, high-level transgene expression in mouse liver, isotonic solutions were injected into mice that had received a hydrodynamic injection of plasmid DNA. Transgene expression in the liver was increased by such injections irrespective of the cDNA, promoter, and type of solution. This reactivation was repeatable and detectable even 3 months after gene transfer. Parameters required for reactivation were similar to those required for the hydrodynamic injection of plasmid DNA. Plasmid DNA-polyethyleneimine complex-based transgene expression in mouse liver was also reactivated by the same treatment. DNA microarray and quantitative RT-PCR analyses revealed that the expression of immediate-early response genes c-fos and c-jun was increased 70 and 100-fold, respectively. Activator protein (AP)-1- or nuclear factor (NF)-kappa B-dependent transgene expression was increased by an injection of isotonic solutions. These findings indicate for the first time that a rapid, large-volume injection of isotonic solution activates the transcription factors AP-1 and NF-kappa B in the liver, which in turn increases the transcription of genes delivered by hydrodynamic injection or other methods.