Based on the hypothesis that tissue macrophages present an obstacle for adenovirus (Ad) vector-mediated gene transfer to internal organs, this study evaluated the consequences of transient depletion of Kupffer cells on subsequent transfer of the Ad vector genome and Ad vector-directed gene expression in the livers of experimental animals. Depletion of Kupffer cells in mice by intravenous administration of multilamellar liposomes containing dichloromethylene-bisphosphonate permitted subsequent intravenous administration of an Ad vector to provide a higher input of recombinant adenoviral DNA to the liver, an absolute increase in transgene expression, and a delayed clearance of the vector DNA and transgene expression. These observations suggest that the tissue macrophages pose a significant hurdle to Ad vector-mediated gene transfer and that strategies to transiently suppress macrophage defenses might be useful in enhancing the efficiency of this in vivo gene transfer system.