Ran GTPase has been shown to be involved in host innate immune response, and two alleles, RanT/n and RanC/d, which differ from each other by a single nucleotide, have opposite effects on host innate immune response. In this study, we showed that although intravenous administration in mice with either Ran cDNA using an identical adenovirus (Ad) vector resulted in no significant difference in vector tissue distribution, intraperitoneal administration resulted in effective vector transduction into peritoneal macrophages, coupled with a striking difference in vector tissue distribution in 2 h or less. We further demonstrated the presence of prepackaged RNA in virions of Ad vectors, in cells actively producing Ad virus particles, and in cells very shortly after Ad infection. Real-time PCR analysis confirmed the presence of prepackaged RNA and estimated the copy number to be one per viral genome. The prepackaged viral mRNA could be used for translation into proteins, as shown by experiments in which the transcriptional inhibitor actinomycin-D was used. Hence, translation of Ran proteins from prepackaged viral mRNA immediately after virus uncoating in the cytoplasm is one mechanism that would account for an early difference in Ad-vector tissue distribution after efficient gene transfer into macrophages.