Most of an intravenous dose of species C adenovirus serotype 5 (Ad5) is destroyed by liver Kupffer cells. In contrast, another species C virus, Ad6, evades these cells to mediate more efficient liver gene delivery. Given that this difference in Kupffer cell interaction is mediated by the hypervariable (HVR) loops of the virus hexon protein, we genetically modified each of the seven HVRs of Ad5 with a cysteine residue to enable conditional blocking of these sites with polyethylene glycol (PEG). We show that these modifications do not affect in vitro virus transduction. In contrast, after intravenous injection, targeted PEGylation at HVRs 1, 2, 5, and 7 increased viral liver transduction up to 20-fold. Elimination or saturation of liver Kupffer cells did not significantly affect this increase in the liver transduction. In vitro, PEGylation blocked uptake of viruses via the Kupffer cell scavenger receptor SRA-II. These data suggest that HVRs 1, 2, 5, and 7 of Ad5 may be involved in Kupffer cell recognition and subsequent destruction. These data also demonstrate that this conditional genetic-chemical mutation strategy is a useful tool for investigating the interactions of viruses with host tissues.