Adenoviral vectors have great potential for use in gene therapy and genetic immunization. The targeting of Ad vectors to the relevant tissue and cell types in vivo could greatly improve their safety and performance by lowering the effective dosage required for therapeutic levels of gene expression. Redirection of Ad vector tropism will require physical modifications of the adenoviral capsid but direct genetic modification of the Ad capsid has so far been limited to small peptides. A novel system for the attachment of targeting ligands to the Ad capsid, based on the extremely strong avidin-biotin interaction, is described herein. The genetic insertion of a biotin acceptor peptide (BAP) into the fiber, protein IX, or hexon components of the Ad capsid has resulted in vectors that are metabolically biotinylated upon production in host cells. Avidin-dependent redirection of transduction through a variety of biotinylated ligands is greatly dependent on the nature of the biotinylated capsid protein. While targeted transduction via the fiber was efficient through a broad array of ligand-receptor interactions, redirection of binding and uptake through the more abundant protein IX and hexon resulted in poor transduction. Although the basis of these differences has not been determined, it most likely reflects functional differences between the capsomeres during the process of vector uptake and trafficking. This study represents the first direct comparison of transduction through the various capsomeres and strongly suggests that future targeting efforts should be focused on fiber modification. In addition to the functional studies on Ad-IX-BAP, structural analysis by cryoelectron microscopy and particle reconstruction is presented. The C-terminal BAP fusion was used as a structural tag to visualize the position of IX within the capsid. Results contradict all previous reports on the location of IX and suggest the surface accessible density currently assigned as IIIa is actually attributable to protein IX. These studies highlight the need for a more thorough analysis of adenoviral structure and the complex interactions between its components.