RNA interference (RNAi) induced by delivery of a small-interfering RNA (siRNA)-expressing vector was characterized in mice. siRNA-expressing plasmid DNA (pDNA) was injected by a hydrodynamics-based procedure along with pDNA encoding an exogenous target luciferase gene. A comparative study showed that stem-loop-type siRNA-expressing pDNA was superior, in terms of the transgene suppressive efficacy, to the tandem-type in the liver following systemic delivery of these pDNAs. Transgene suppression occurred in the liver, kidney, and lung as well as muscle. The degree of suppression was dependent on the dose of siRNA-expressing pDNA and the time at which transgene expression was determined following simultaneous injection of siRNA-expressing and target pDNAs. A reduction in transgene expression became apparent at 1 day after injection, whereas a lower degree of inhibition was obtained before this, as early as 6 h even in mice treated with an excess of siRNA-expressing pDNA. These results suggest that delivery of siRNA-expressing pDNA requires a period of time for induction of RNAi. A study of sequential injections revealed that prior injection of siRNA-expressing pDNA produced a significant suppression for at least 1 day, which disappeared within 4 days. Confocal microscopic studies indicated that the localization of the cells with successful delivery of transgene was different between primary and secondary hydrodynamics-based injections, accounting for the less effective inhibition following the sequential injections. Taken together, these results demonstrate that vector-based in vivo RNAi is a dose- and time-dependent process and offers the possibility of suppressing endogenous targets in a variety of somatic cells.