Targeting adenovirus vectors (AdV's) for selective transduction of specific cell types requires ablation of native adenovirus tropism and introduction of a unique target-binding moiety. To bring these requirements within reach, we developed a novel strategy to target AdV's genetically that relies on replacement of the entire adenovirus fiber protein with a fusion molecule comprising the virion-anchoring domain of fiber and the oligomerization domain of reovirus attachment protein sigma1. The chimeric molecule forms trimers, is transported to the nucleus, and assembles onto the adenovirus capsid. In contrast to previously reported genetically targeted vectors, the AdV presented herein propagates efficiently without a requirement for complementing fiber. Due to ablation of the native adenovirus tropism, the infectivity of this AdV was at least 35-fold reduced on 293 cells. Importantly, a His tag incorporated into the chimeric attachment protein conferred His-tag-dependent tropism to the AdV, which resulted in a 12- to 40-fold greater transduction efficiency on two different cell lines expressing a His-tag-binding receptor. In addition, the infection efficiency was strongly reduced by preincubation with a His-tag-specific Ab. Thus, this sigma1-based chimeric attachment molecule provides a promising new platform for the generation of truly targeted AdV's.